Therapeutic and diagnostic methods using total leukocyte surface antigens

ABSTRACT

The invention relates to the measurement of total leukocyte antigens, or fragments thereof, and the use of such measurements to enumerate cells, especially in whole blood. The term &#34;total&#34; leukocyte antigen used herein refers to the total amount of a leukocyte antigen in a sample, including that present in membrane and intracellular compartments and extracellular soluble compartments. Measurements of a total leukocyte antigen can be used to type cells, detect or diagnose disease or to monitor disease therapy. In a further embodiment, the invention relates to the measurement of both the amount of total leukocyte antigen and the amount of the soluble form of the leukocyte antigen and a comparison of the measured levels.

1. INTRODUCTION

The present invention is directed to the measurement of total leukocytesurface antigens, such as T cell receptors and T cell differentiationantigens or fragments thereof, and the application of such measurementsin the diagnosis and therapy of diseases and disorders. The presentinvention is also directed to the measurement of soluble leukocyteantigens or fragments thereof, in conjunction with the measurement oftotal leukocyte surface antigens and the application of such dualmeasurements in the diagnosis and therapy of diseases and disorders. Themeasurement of such molecules, and preferably a plurality of suchmolecules, can be used in monitoring the effect of a therapeutictreatment, detecting and/or diagnosing disease.

2. BACKGROUND OF THE INVENTION 2.1. LEUKOCYTE SURFACE MOLECULES

Clusters of differentiation (CD) have been established which definehuman leukocyte differentiation antigens (Bernard and Boumsell, 1984,Hum. Immunol. 11:1-10; Knapp et al., 1989, Immunol. Today 10:253:258;Gebel et al., 1988, ASHI Quarterly 12:11; Leukocyte Typing III: WhiteCell Differentiation Antigens. Ed., McMichael, A. J. 1987. OxfordUniversity Press. Oxford), by the comparison of reactivities ofmonoclonal antibodies directed against the differentiation antigens. TheT cell surface antigens, their classification into epitope-definedsubgroups, and their distributions on T cells have been studied by useof monoclonal antibodies directed against human T cells (Clark et al.,1983, Immunogenetics 18:599-615; Hansen et al., 1984, in LeucocyteTyping, Bernard, A., et al., eds., Springer-Verlag, New York, pp.195-212). Some of the T cell clusters of differentiation and other cellsurface molecules are listed in Table I.

These cell surface markers serve as markers of cell lineage, theidentity of the functional T cell subset to which the T cell belongs,and the activation state of the T cell. Several of the cell surfacemolecules have been studied in great detail, have been found to beimportant in initiating and regulating immune functions and are criticalto communication processes between immune cells.

                  TABLE I                                                         ______________________________________                                        LEUKOCYTE SURFACE MOLECULES                                                   Cell                    Detection                                             Surface                 Monoclonal  Refer-                                    Marker     Expression   Antibodies  ences                                     ______________________________________                                        T cell Antigen                                                                           All T cells and                                                                            T40/25,     1, 2, 3, 4,                               Receptor   T cell subsets                                                                             αF1, βF1,                                                                      5, 6                                                              δTCS1,                                                                  TCRδ1,CγM1                                CD1        Thymocytes & OKT6                                                             Langerhans   NAI/34                                                           Calls, Lukemia                                                                Cells                                                              NK cell    NK cells     NC-37       7                                         receptor                specific                                                                      antibodies                                            Cell Adhesion                                                                 Molecules                                                                     CD2        All T cells  OKT11       8, 9, 10                                                          Leu5                                                                          B67.1                                                 CD58 (LFA-3)                                                                             Leukocytes,  TS2/9       11                                                   epithelial                                                         CD3        Pan T cell   OKT3        12                                                                Leu4                                                  CD4        Helper/Inducer                                                                             OKT4        12                                                   Subsets of T Leu3a                                                            cells                                                              CD5        T.B subsets  UCHT2       11                                                                T1                                                    CD7        T Cells      3Al         11                                        CD8        Supressor/   α Chain:                                                                            13, 14                                               Cytotoxic    OKT8                                                             Subsets of T Leu2a                                                            cells        β Chain:                                                                             14a                                                               T8/2T8                                                β2 integrins                                                             LeuCAM     leukocyte cell           11                                                   adhesion                                                                      molecules                                                          CD11a (LFA-1)                                                                            myeloid,                 11, 15                                               lymphoid                                                           CD11b      myeloid                  11, 15,                                   (MAC-1 (CR3))                       16, 17,                                                                       18, 19                                    CD11c (CR4)                                                                              myeloid                  11, 15                                    CD16 (FcR111)                                                                            Natural Killer,                                                                            HUNK2       11                                                   Macrophages  3G8                                                              Granulocytes                                                       CD21 (CR2) B subset     B2          11, 20                                                            HB5                                                   CD23 (FCεR11)                                                                    B subset     MHM6        11                                                                Blast-2                                               CD25       TAC IL-2     Anti-TAC    21                                                   Receptor     7G7/B6                                                           (Activated T                                                                  Cells)                                                             CD30       Activated T, B                                                                             Ki-1        11                                                   Cells        HSR4                                                             Reed-Steinberg                                                                Cells                                                              CD35 (CR1) Granulocytes, B                                                                            YZ-1        11, 22                                               cells, monocytes                                                                           J3D3                                                  β3 integrins                                                             CD41                                11, 23                                    CD51                                11, 23                                    Homing     Leukocytes,  33-3B3      24                                        Receptors  brain        GRHL1       25, 26                                    CD44                                                                          Mel-14                                                                        β1 integrins                                                             CD49a-f(VLA-1)                                                                           Extra cellular           27, 23                                    VLA-2, VLA-3,                                                                            Matrix (ECM)                                                       VLA-4                                                                         CD56       Natural Killer                                                                             NKH1        11                                        (NKH1, NCAM)                                                                             Activated    Leu19                                                            lymphocytes                                                        CD71       Transferring             11, 28                                               Receptor,                                                                     Proliferating                                                                 cells                                                              ______________________________________                                         List of References for Table I                                                1 Brenner et al., 1984, J. Exp. Med. 160:541-551.                             2 Henry et al., 1989, Hybridoma, 8:577.                                       3 Brenner et al., 1987, J. Immunol., 138:1502.                                4 Wu et al., 1988, J. Immunol., 141:1476.                                     5 Band et al., 1987, Science 238:682.                                         6 Hochstenbach et al., 1988, J. Exp. Med. 168:761.                            7 Evans, International patent publications #W089/03394, #W088/03395, &        #W088/03396 published April 20, 1989                                          8 Bierer et al., 1989, Annu. Rev. Immunol. 7:579-99.                          9 Verbi et al., 1982, Eur. J. Immunol. 12:81-86.                              10 Perussia et al., 1983, J. Immunol. 133:180.                                11 Knapp et al., 1989, Immunol. Today 10:253.                                 12 Kung et al., 1979, Science 206:347-349.                                    13 Reinherz et al., 1979, Proc. Natl. Acad Sci. USA 76:4061-4065.             14 Ledbetter et al., 1981, Monoclonal Antibodies and Tcell Hybridoma,         Elsevier, North Holland, N.Y. pp 16-22.                                       14a Shieu, L., et al., 1988, J. Exp. Med. 168(6):1993-2005.                   15 Kishimoto et al., 1989, Adv. Immunol. 46:149-182.                          16 Altieri & Edgington, 1988, J. Biol. Chem 263:7007-1                        17 Wright et al., 1988, Proc. Natl..Acad. Sci. USA 85:7734-38.                18 Wright et al., 1989, J. Exp. Med. 169:175-83.                              19 Russell & Wright, 1988, J. Exp. Med. 168:279-92.                           20 Nemerow et al., 1985, J. Virol. 55:347-351.                                21 Uchiyawa et al., 1981, J. Immunol. 126(4):1393-1397.                       22 Klickstein et al., J. Exp. Med. 165:1095-1112.                             23 Hemler, 1990, Annu. Rev. Immunol. 8:365-400.                               24 Berg et al., 1989, Immunol. Rev. 108:1-18.                                 25 Lasky et al., 1989, Cell 56:1045-55.                                       26 Haynes et al., 1989, Immunol. Today 10:423.                                27. Hynes, 1987, Cell 48:549.                                                 28. Reinherz et al., 1980, Proc. Natl. Acad. Sci. USA, 77:1588-1592.     

2.2. LEUKOCYTE SURFACE MOLECULE SPECIFIC ANTIBODIES

Over the last several years, antibodies to determinants of murine andhuman TCRs have been developed. Some of these antibodies appear torecognize all members of a V region family, some a subset of V regionswithin a family, and some a particular V region only (TABLE II). TheseTCR antibodies identify minor populations of peripheral blood T cells(1-5%) and subdivide T cells in a new way based on TCR V region usage.

                  TABLE II                                                        ______________________________________                                        Name   Clone   Specificity                                                                              Immunogen                                                                             Reactivity                                                                            Ref*                                ______________________________________                                        βV5(a)                                                                          1C1     Vβ5.2 and                                                                           HPB     1-5%    1                                                  Vβ5.3         of PBL                                                     Subfamilies                                                    βV5(b)                                                                          W112    Vβ5.3 HPB     0-3%    2                                                  Subfamily          of PBL                                                     subset of                                                                     βV5(a)                                                    βV8(a)                                                                          16G8    Vβ8 family                                                                          JURKAT  1-5%    2                                                                     of PBL                                      βV12(a)                                                                         S511    Vβ12 family                                                                         SEZARY  1-5%    3                                                                     of PBL                                      βV6(a)                                                                          OT145   Vβ6 family                                                                          T-CLL   0-5%    4                                                  Allotypic          of PBL                                                     Vβ6.7                                                                    epitope                                                        αV2(a)                                                                         F1      Vα2  T-CLL   1-5%    5                                                  Subfamily          of PBL                                      αβV(a)                                                                    LC4     Vβ5.1 SUP-T13 1-5%    6                                                  Subfamily          of PBL                                      ______________________________________                                         List of References for Table II                                               1 Boylston et al., 1986, J. Immunol. 137:741-744.                             2 Tian et al., 1989, FASEB J. 3:A486 Abstr.                                   3 Bigler et al., 1983, J. Exp. Med. 158:1000-1005.                            4 Posnett et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:7888-7892.           5 Janson et al., 1989, Canc. Immunol. Immunother. 28:225-232.                 6 Maecker & Levy, 1989, 1989 J. Immunol. 142:1395-1404.                  

These reagents have proven valuable in studying the repertoire of TCRsexpressed under many in vivo and in vitro conditions. For example, invitro stimulation of mononuclear cells with certain bacterialenterotoxins (superantigens) leads to the expansion of T cellsexpressing a limited number of TCR Vβ families (Kappler et al., 1989,Science 244:811-813). In vivo expansion of T cells bearing particular Vβregions has been detected in certain animal models of autoimmunediseases.

Other antibodies recognize common determinants on all αβ or γδ TCRs.These include CγM1, a monoclonal antibody which has been shown tospecifically recognize the TCR-γ protein (Hochstenbach et al., 1988, J.Exp. Med. 168:761). This monoclonal antibody was generated against aconstant-region encoded peptide and reacts with both Cγ1 and Cγ2 encodedTCR-γ chains. It appears to possess framework reactivity against allTCR-γ polypeptides.

TCRδ1 (Band et al., 1987, Science 238:682) and δTCS1 (Wu et al., 1988,J. Immunol. 141:1476) are monoclonal antibodies specific for the δ chainof the human δγ TCR. Unlike clone specific or idiotypic anti-TCRantibodies, TCRδ1 appears to identify all T cells which express the γδTCR. δTCS1 identifies a minor subset of these γδ T cells.

Additional monoclonal antibodies to the T cell receptor gamma and deltachains have also been reported (European Patent Publication #EP289252Published Nov. 2, 1988; International Patent Publication #WO88/00209published Jan. 14, 1988).

βFI (Brenner et al., 1987, J. Immunol. 138:1502-1509) is a murinemonoclonal antibody specific for a framework, i.e., common ornonpolymorphic determinant on the β chain of the αβ TCR and identifiesall T cells expressing the αβ TCR. αF1 (Henry et al., 1989, Hybridoma8:577) is a monoclonal antibody specific for a framework determinant ofthe α chain and identifies all T cells expressing the αβ TCR.

Antibodies to CD4 have been widely described (Kung, P. C., et al., 1979,Science 206:347-349) and are commercially available. A series of suchantibodies reacting with non-competing epitopes on the CD4 molecule havebeen described. Such a set has been termed OKT4, OKT4A, OKT4B, OKT4C,OKT4D, OKT4E, and OKT4F (Rao, P. E., et al., 1983, Cell. Immunol.80:310).

Antibodies directed against the CD4 or CD8 antigens have been shown toblock cell function. Antibodies against CD4 block most helper Tfunctions, mixed lymphocyte reactions and induction of T helper activity(Biddison et al., 1984, J. Exp. Med. 159:783). Antibodies against CD8block the cytotoxic activity of CD8 positive cytotoxic T lymphocytes(Swain, S. L., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:7101-7105).Antibodies against CD4 have also been described that are capable ofactivating CD4-positive T cells. CD4 is internalized upon treatment ofthe cells with phorbol esters and resulting phosphorylation (Hoxie, J.A., et al., 1986, J. Immunol. 137:1194-1201).

Antibodies to CD35 (Wong et al., 1985, J. Immunol. Methods 82:303; Yoon& Fearon, 1985, 134:3332; Schrieber, U.S. Pat. No. 4,672,044 issued Jun.9, 1987) have also been reported.

2.3. CLINICAL APPLICATIONS

These various lymphocyte cell surface markers have enormous clinicalapplication potentials for the identification of lymphocyte populationsand their functional status (Krensky, A. M. and Clayberger, C., 1985,Transplant. 39 (4):339-348; Kung, P. C., et al., 1984, MonoclonalAntibodies in Clinical Investigations, ClinicalBiochemistry-Contemporary Theories and Techniques, Vol. 3, AcademicPress, pp. 89-115; Kung, P. C., et al., 1983, Int. J. Dermatol.22.(2):67-733; Cosimi et al., 1981, N. Engl. J. of Med. 305:308; Knowleset al., 1983, Diagnostic Immunol. 1:142; Hoffman, 1984, Amer.Biotechnol. Lab 2:39).

Existing clinical methods of T cell typing involve the use of monoclonalantibodies which define T cell surface markers to detect the presence ofspecific cell surface markers on the T cell surface. Measuring the totalnumbers of T cells expressing a marker on the surface or membrane hasbeen useful for the characterization and classification of lymphoidmalignancies (Greaves, M., et al., 1981, Int. J. Immunopharmac. 3(3):283-300). Changes in the relative percentage of T helper and Tsuppressor/cytotoxic cells were found to be associated with immuneevents in renal transplantation due to viral infection (Colvin, R. B.,et al., 1981, Proc. 8th Int. Congr. Nephrol., Athens, pp. 990-996),autoimmune diseases (Veys, E. M., et al., 1981, Int. J. Immunopharmac.3(3):313-319), and AIDS (Gupta, S., 1986, Clin. Immunol. Immunopathal.38:93-100; Ebert, E. C., et al., 1985, Clin. Immunol. Immunopathol.37-283-297).

The expression of T cell surface markers has also been used for theassessment of the immune status of patients. It has been establishedthat by measuring the relative number of distinct, functional T cellsubsets, and/or the relative number of activated T cells in peripheralblood or tissues, an assessment of the immunological condition of apatient is possible.

In an HIV-infected individual, the most useful single prognosticindicator for progression to overt AIDS is the absolute number of CD4⁺ Tcells/mm³ of whole blood. Currently, accurate measurement of CD4⁺ Tcells requires the use of a flow cytometer, both costly and not widelyavailable. In addition, federal guidelines define eligibility for AZTadministration to AIDS infected individuals solely on the basis of theCD4⁺ T cell count (a T cell count which drops below 500 CD4⁺ Tcells/mm³) regardless of symptomology. The cell count cut off forpentamidine treatment is 200 cells/mm³. However, the costly and largelyunavailable flow cytometer leaves a large population of clinicians andpatients without the means of proper immune status evaluation andtherefore AZT treatment.

If the CD4⁺ T cell count drops below 500 cells/mm³ (AZT treatment) orbelow 200 cells/mm³ (pentamidine treatment) (Cowley et al, Jun. 25,1990, Newsweek, pp. 23-27; Mills & Masur, August, 1990, ScientificAmerican, pp. 50-57; Fahey et al., 1990, New Engl. J. Med. 322:166-72;Goedert et al., 1989, New Engl. J. Med. 321:1141-8), then the patient isindicated for therapeutic intervention.

The measurement of CD4 positive or CD8 positive T cells may be used todetect or diagnose disease, or monitor disease treatment as described inpatents and publications U.S. Pat. Nos. 4,709,015, 4,725,543, 4,361,550,4,908,203, WO91/10722, WO89/08143, WO91/07985, EP 421,380, WO91/09966,WO91/03493, EP 403,935, WO88/04327, U.S. Pat. Nos. 4,695,459, and4,649,106.

2.4. METHODS OF MEASURING LEUKOCYTE SURFACE MOLECULES IN A SAMPLE

The methods for detecting, staging or diagnosing a disease, ormonitoring the progress of therapy of a disease discussed above requireseparation of the components of a sample.

For example, methods of diagnosis or monitoring of a patient with adisease may depend on detection of the amount of a soluble leukocyteantigen, and comparison of the amount of soluble leukocyte antigen inthe sample from a patient to the amount in a sample from a normalindividual or in the same individual at an earlier time. See, e.g.,publications WO 87/05912, and WO 90/04180. However, the assay samplemust be isolated from cells and cellular debris, which is timeconsuming.

Prior to the instant invention, measurement of the amount of leukocytespositive for a leukocyte marker was carried out by direct analysis ofcells. To date, investigators have primarily measured the amount of cellsurface markers in enriched cell populations derived from whole blood.This involved separating whole blood into its serum (or plasma) and cellconstituents followed by enrichment of the desired cells by proceduressuch as those involving the lysis of red blood cells and subsequentisolation of white blood cells on density gradients of polymers, such asFicoll-Hypaque. The enriched cell populations can then be analyzedeither by direct or indirect immunofluorescence involving flowcytometers or fluorescent microscopes, or alternatively lysed withappropriate buffers followed by analysis of either the total lysate ormembrane and cytosolic components individually. Limitations of theseprocedures include (1) the requirement for fresh samples, (2) the needto use enriched cell populations rather than whole blood, (3) therequirements of expensive equipment, (4) the time involved in preparingthe samples and (5) the need for fairly large sample sizes or cellnumbers for analysis, since cells are lost during the sample preparationsteps and because flow cytometric analyses require that a statisticallysignificant number of cells be analyzed for reliable measurements to beobtained. In diseases where the cells of interest are steadilydeclining, (for example, the decrease in the number of CD4 cells duringHIV infection and progression to AIDS) larger sample volumes must beused in order to obtain a significant number of cells to analyze in theenriched cell population.

3. SUMMARY OF THE INVENTION

The present invention overcomes the deficiencies of the prior methods ofdetecting leukocyte antigens and of estimating the number of cellspositive for a particular leukocyte marker by providing a reliable,quantitative, easy-to-use method to measure total amount of a leukocytemarker in a sample containing cells in the biological fluid in which thecells are obtained. Alternately, the sample can contain just cells.

Surprisingly, it has been discovered that treatment of cells in a samplewith concentrated detergent followed by dilution of the samplesolubilizes the total amount of leukocyte markers in the sample. Thus,the method for determining the total amount of a leukocyte marker in asample comprises adding a concentrated non-ionic detergent solution tothe sample to form a detergent-treated sample, and allowing thedetergent to lyse the cells, and/or disrupt membranes. Having releasedany intracellular and/or membrane bound marker in this manner, the totalamount (i.e., inclusive of marker so released as well as any solublemarker that may be present originally in the sample) of leukocyte markerin the sample can be detected. After the cells have been lysed, thesample is diluted to reduce the detergent concentration. Detection ofthe amount of leukocyte marker in the sample is by immunologicaldetection means, e.g., immunoassay.

Remarkably, measurements of total leukocyte markers can be used todetermine the approximate number of leukocytes positive for theleukocyte marker, in a sample i.e., measurement of total leukocytemarker can be used to enumerate cells expressing that antigen with anappropriate correlation. Even more remarkable is the linearity of thecorrelation between the measurements obtained according to the method ofthe invention and cell counts achieved by conventional means. Hence, theinstant invention represents a highly advantageous substitute for themore cumbersome methods of the prior art.

Measurements of total leukocyte markers are useful in monitoring theeffectiveness of a treatment of a subject, in predicting therapeuticoutcome or disease prognosis, and in evaluating and monitoring theimmune status of patients. Measurements of total leukocyte markers canbe accomplished by sandwich enzyme immunoassays where the samples aretreated so that the total amount of a leukocyte marker present inmembrane, intracytoplasmic and soluble compartments can be measured.

In particular embodiments, the invention is directed to the measurementof amounts of total CD4 antigen. Total CD4 antigen measurements areparticularly useful in diseases where the absolute number of CD4⁺ cellsis the best indicator of disease prognosis or treatment outcome. Suchdiseases include, but are not limited to, AIDS.

In another embodiment, the invention directed to the measurement of thetotal amount of CD8 antigen. Total CD8 antigen measurements areespecially useful in diseases associated with modulation of the CD8⁺subset of leukocytes. These include but are not limited to infectiousdisease, autoimmune disease, and transplantation rejection illness.

In yet another embodiment, the invention is directed to the measurementof the total amount of T cell antigen receptor (TCR) present in asample. Total TCR measurements can include the measurement of total TCRon all cells expressing any αβ or γδ TCR or can include the measurementof total TCR on particular subsets of cells including, but not limitedto, subsets expressing specific Vα, Vβ, Vγ and/or Vδ peptides.

In a further embodiment, the invention is directed to the measurement ofboth the amount of total leukocyte marker and the amount of the samesoluble leukocyte marker and a comparison of the measured levels. Thechange in the total levels and soluble levels relative to one anotherduring disease progression or disease treatment can be superior to themeasurement of either total or soluble levels alone. Such measurementsare useful for the detection, diagnosis or monitoring of treatment of adisease or disorder.

In another embodiment, the invention provides for the measurement of thetotal amount of two or more leukocyte markers, and comparison of theamounts of the markers. The relative amounts of the markers, or timedependent variation in the relative amounts of the markers duringdisease progression, can be used to detect, diagnose, or monitortreatment or progress of a disease or disorder. Significantly, themeasurement of total leukocyte markers can be used to estimate thenumber of cells positive for each marker. The ratios of total leukocytemarker or approximate numbers of cells positive for each leukocytemarker can be compared with the same ratios in a normal individual, andthis comparison used to detect, diagnose, or stage a disease or monitortreatment of a disease or disorder.

Simultaneous measurement of the total amount of a marker is animprovement over separately measuring the cell bound, cell lysate orsoluble marker for the following reasons. Firstly, the measurement caninclude the total amount of markers present in all three compartments,not just the amounts present in one or two compartments. Secondly, themeasurement of total markers is easier than other procedures thatinvolve greater sample preparation, complex equipment and more steps.Thirdly, small quantities of sample, e.g., 100 μl, and as little as 5-10μl of whole blood, can be directly analyzed in a simple immunoassayformat without prior enrichment of the samples. The small volume ofwhole blood necessary has major benefits in pediatric applications withinfants and small children. This represents a significant cost reductionper sample analyzed, and the elimination of an expensive equipmentrequirement, thereby making the analysis widely available to manylaboratories or clinics. Fourthly, the measurement of total marker is animprovement, since it involves minimum sample preparation and does notcreate aerosols that are hazardous in the case of infectious samplesfrom patients. Most important, for infectious samples such as thosecontaining HIV, the solubilization procedure, i.e., treatment withconcentrated detergent, inactivates the virus, thereby making subsequentanalysis safer. Fifthly, the total marker assay does not require freshsamples. Each patient sample can be treated and stored frozen. This isespecially useful for a series of samples obtained from the same patientover a period of time as in a longitudinal study. Each sample canquickly be treated and frozen so that all samples can be thawed andanalyzed simultaneously. This is a definite improvement over flowcytometric analysis where the cells need to be fresh and intact. It alsoeliminates variance obtained in assay results that arise from interassayvariability, since all of the assays may be performed at one time.

3.1. DEFINITIONS

As used herein, the following terms will have the meanings indicated:

    ______________________________________                                        Total Leukocyte = the total amount of a                                       Marker            leukocyte antigen or                                                          fragment thereof                                                              (including                                                                    membrane-associated,                                                          intra- and extracellular)                                                     present in a sample.                                        WBCC =            White blood cell count                                      AZT =             azido-deoxthymidine                                         HTLV III/LAV/HIV =                                                                              Human T cell Leukemia                                                         Virus Type                                                                    I/Lymphadenopathy                                                             Associated Virus/Human                                                        Immunodeficiency Virus                                      OPD =             O-phenylenediamine                                          mAb =             monoclonal antibody                                         Spontaneous release =                                                                           release by normal or                                                          pathologic physiological                                                      processes of the cell                                       AIDS =            Acquired immunodeficiency                                                     disease syndrome                                            TCAR =            T cell antigen receptor                                     ______________________________________                                    

4. DESCRIPTION OF THE FIGURES

FIG. 1. The measurement of total CD4 antigen in whole blood according tothe method of the invention using a CELLFREE® CD4 assay (see Section5.1, Supra). The three curves represent the detection of total CD4 (O.D.490) from the blood of three normal individuals.

FIG. 2. The correlation between total CD4 antigen as measured in a CD4immunoassay according to the method of invention and the total number ofCD4 positive cells/mm³ of blood. Total CD4 (O.D. 490) was measured from2.5 μl of whole blood from five different normal individuals using aCELLFREE® CD4 assay. The number of CD4 positive cells/mm³ was measuredin the blood samples three days after determination of total CD4 antigenfrom the samples. Thus, the number of total CD4⁺ cells/mm³ of bloodappear lower than expected for normal individuals. However, the curveobtained shows a linear relationship between total CD4 antigen and thenumber of CD4+ cells.

FIG. 3. The correlation between total CD4 antigen measured from wholeblood of HIV-infected individuals and one normal control and total CD4⁺cells/mm³ of blood. HIV-infected individuals are represented by a singlesquare and the normals by the double square.

FIG. 4. The correlation between total CD4 antigen measured from wholeblood of HIV-infected individuals and normal controls and total CD4⁺cells/mm³ of blood. HIV-infected individuals are represented by a singlesquare and the normal by the double squares.

FIG. 5. Total CD4 antigen method standard curve.

FIG. 6. Comparison of the ability to enumerate CD4 positive cells by thetotal CD4 antigen method and by flow cytometry in 95 normal and diseasesamples.

FIG. 7. Comparison of the ability to enumerate CD8 positive cells by thetotal CD8 antigen method and by flow cytometry in 86 normal and diseasesamples.

FIG. 8. Cell associated CD8 (total CD8 antigen minus soluble CD8antigen) in whole blood of 30 normal and 11 cancer patients.

FIG. 9. Total T cell antigen receptor β chain antigen in whole blood ofa normal healthy donor.

FIG. 10. Total T cell antigen receptor Vδ1 chain antigen and immunoassaystandard curve.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the measurement of total leukocytesurface antigens, total T cell differentiation antigens, or relatedtotal markers or fragments thereof, and the use of such measurements inthe diagnosis and therapy of diseases and disorders.

The measurement of total markers according to the invention is valuablein cell enumeration, cell typing, monitoring the effect of a therapeutictreatment on a subject, detecting and/or diagnosing a disease in asubject, in predicting therapeutic outcome or disease prognosis and inevaluating and monitoring immune status of patients. A plurality oftotal markers can be measured, such as total CD4 antigen and total CD8antigen. In a preferred embodiment in relation to AIDS, assays areconfigured such that total CD4 and total CD8 antigens can be measuredsimultaneously.

As used herein, the term "total" shall mean the total amount of themarker present in the sample. For example, in a particular sample suchas a sample that comprises whole blood, the total marker includes theamount of marker present in the cell membrane, intracytoplasmic andsoluble serum compartments. The soluble compartment can include bothspontaneously released soluble marker as well as soluble recombinantmarkers that may have been administered as a therapeutic treatment. Inanother sample, such as in a sample that comprises cells in tissueculture, the total marker includes the amount of marker present in themembrane, intracytoplasmic and cell culture media compartments of thesample. In yet another embodiment in which the sample comprises atissue, e.g., a biopsy specimen, the total marker includes the amount ofmarker present in the membrane, intracytoplasmic and interstitialcompartments.

As used herein, the term "compartment" modified by membrane,intracytoplasmic, and soluble elements of a sample refers to the totalamount of all membrane, the total amount of intracytoplasmic contents,and the total amount of soluble material included in the sample.

As used herein, the term "leukocyte marker" refers to an antigen orpolypeptide found on the cell surface of a leukocyte. Such markersinclude but are not limited to cell surface antigens, CD (clusters ofdifferentiation) antigens, receptors, or other cell surface polypeptidesor proteins. The term leukocyte marker is further intended to include animmunologicaly active fragment, e.g., an epitope, of the foregoingmolecules. The leukocyte markers for detection according to the presentinvention, and the particular cells known to express these markers, aresummarized in Table I in Section 2.1, supra. In specific embodiments,the leukocyte marker can be CD4, CD8 or T cell antigen receptor (TCAR).

As used herein, the term "leukocyte" refers to the usual meaning of thatterms, i.e., immune associated cells derived from hematopoietic stemcells, such as lymphocytes (B cells and T cells) and myeloid lineagecells (neutrophils, macrophages, eosinophils, megakaryocytes,erythrocytes, mast cells).

As used herein, a "sample" refers to a collection of cells in themilieux in which they were obtained, i.e., a biological fluid, or tomembrane and/or intracytoplasmic components of the cells. Totalleukocyte antigens may be measured in samples derived from a biologicalfluid, e.g., whole blood, plasma, serum, blood cells, saliva, urine,synovial fluid, pleural effusions, tumor and tissue infiltrates,amniotic fluid, spinal fluid or cranial fluid. In another embodiment,the biological fluid may be cell culture fluid. The sample can comprisetissue, including interstitial fluid. Preferably when the sample is atissue sample, the tissue is treated to disrupt the connective tissuematrix, e.g., by trypsin digestion or homogenization. In anotherembodiment, the sample comprises cells derived from the foregoingsources.

5.1. DETECTION AND MEASUREMENT OF TOTAL LEUKOCYTE MARKER

The total amount of a leukocyte marker exists in three compartments: themembrane, intracytoplasmic and released/soluble compartments ("totalleukocyte marker"). The invention includes immunoassays thatsimultaneously measure the total amount of a leukocyte marker present inall three compartments or in the membrane and/or intracytoplasmiscompartment, depending upon how the sample is prepared. To determine thetotal amount of a leukocyte marker, an "original" sample, such as wholeblood or blood cells, is first treated to solubilize the cellularcomponents (step 1). The preferred method of solubilizing the cells inthe sample without interfering with immunospecific binding is to treatthe cells with a concentrated non-ionic detergent or detergents to lysethe cells efficiently, thus forming a "detergent-treated" sample. Afterthe cells have been lysed the detergent-treated sample is diluted priorto assay.

Non-ionic detergents for use in present invention include but are notlimited to TRITON® X-100, NONIDET® P-40 (NP-40), Tween-20(polyoxyethylenesorbitan), CHAPS(3-[(3-cholamidopropyl)-dimethylammoniol]-1-propane-sulfonate), tomention a few. In a preferred embodiment, cells are solubilized withTRITON® X-100, NONIDET® P-40, Tween-20 and/or CHAPS. In a more preferredembodiment, cells are solubilized with concentrated detergent to give afinal detergent concentration of about 2% to about 4% in thedetergent-treated sample.

According to the invention, the total volume of non-ionic detergentadded to the sample should not dilute out any leukocyte marker in thesample. Preferably, the volume of detergent does not exceed about 25% ofthe sample volume; more preferably, it does not exceed about 20%. Thenon-ionic detergent or detergents can be added to the original sampleneat, or they can be prepared in a concentrated solution. In addition tothe non-ionic detergent or detergents, the concentrated detergentsolution can comprise distilled water or buffer. The concentration ofnon-ionic detergent or detergents in the solution will preferably be 5times, and more preferably 6 times the final concentration of thenon-ionic detergent or detergents after addition to the sample, i.e., inthe detergent-treated sample.

In an even more preferred embodiment, more than one non-ionic detergentis used to lyse the cells. For example, a high concentration of Tween-20and TRITON® X-100, or TRITON® X-100 and NONIDET® P-40, or NP-40 andTween-20 can be used. The concentration of each detergent ranges fromabout 1% to about 2% after addition to the sample; the totalconcentration of detergent in the detergent-treated sample ranges fromabout 2% to about 4%. More preferably the total detergent concentrationranges from about 2% to about 3%; and even more preferably from about 2%to about 2.5%. In a specific embodiment, the final detergentconcentration in the sample is 1.5% TRITON® X-100 and 1% "NONIDET®"P-40. This is concentration preferred if both CD4 and CD8 are to beassayed.

In another embodiment, the detergent concentration is a concentrationthat inactivates virus, especially HIV. It is a particular advantage ofthe invention that the lytic concentration of non-ionic detergent, suchas the preferred ranges set forth above, is also a virus-inactivatingconcentration.

Other methods of solubilizing cells, e.g., repeated freeze-thaw cycles,sonication, hypotony, or the addition of lower concentrations ofdetergents, are not as effective. Ionic detergents such as SDS are noteffective, since SDS interferes with the subsequent antibody-antigenbinding.

After solubilization for at least about one minute, preferably for aboutone to about five minutes, the sample is diluted with buffer (step 2)prior to analysis in an immunoassay. The dilution can be 2-fold;preferably it is 5-fold or greater. The buffer is chosen to becompatable with the immunoassay for detecting the leukocyte antigen.Buffers preferred for various immunoassays are well known in the art. Ina specific embodiment, sample buffer is 1% bovine serum albumin, 0.25%"NONIDET®" p-40 and 0.01% thimerosal in phosphate buffered saline (PBS).

The present method is not limited by the amount of sample available. Inone embodiment, about 100 μl of a detergent-treated sample of wholeblood may be assayed for total leukocyte antigen. In a more preferredembodiment, about 2.5 to about 25 μl of a detergent-treated sample ofwhole blood may be used. Similar amounts of a sample of culturesuspension, pleural effusion, or other biological fluid can be used. Theactual amount of sample assayed can be varied by adjusting the dilutionfactor. Determination of specific parameters can be accomplished by asimple dilution series assay, and is well within the level of ordinaryskill in the art.

Furthermore, a solubilized sample may be stored frozen so that samplestaken at different times may be assayed in a single experiment. In oneembodiment, the sample is stored at about -20° C. In a preferredembodiment, the sample is stored at about -70° C.

Any method of detecting and measuring leukocyte antigens may be used inthe practice of this invention. Such methods include but are not limitedto competitive and non-competitive assay systems using techniques suchas radioimmunoassays, ELISA (enzyme linked immunosorbent assay),"sandwich" immunoassays, immunoradiometric assays, fluorescentimmunoassays, and protein A immunoassays, to name but a few. U.S. Pat.No. 4,845,026, issued Jul. 4, 1989, entitled "Assay Systems forDetecting Cell-Free T Cell Antigen Receptor Related Molecules andClinical Utilities of the Assays" and U.S. Pat. No. 5,006,459, issuedApr. 9, 1991, entitled "Therapeutic and Diagnostic Methods Using SolubleT Cell Surface Molecules" teach preferred methods of immunoassay. Theirteachings are incorporated herein by reference.

In a preferred embodiment, a sandwich enzyme immunoassay can be used.One description of such an embodiment follows: An antibody (captureantibody, Ab1) directed against the leukocyte marker is adsorbed onto asolid substratum. The leukocyte marker present in the sample binds tothe antibody, and unreacted sample components are removed by washing. Anenzyme-conjugated antibody (detection antibody, Ab2) directed against asecond epitope of the leukocyte marker binds to the antigen captured bymAb1 and completes the sandwich. After removal of unbound Ab2 bywashing, a substrate solution is added to the wells. A colored productis formed in proportion to the amount of antigens present in the sample.The reaction is terminated by addition of stop solution and absorbanceis measured with a spectrophotometer. A standard curve can be preparedfrom known concentrations of the leukocyte marker, from which unknownsample values can be determined.

In a preferred embodiment for the measurement of total CD8 antigenlevels, anti-CD8 mAbs 4C9 and 5F4 can be used as the capture anddetection antibodies, respectively, in a sandwich enzyme immunoassay; ina more preferred embodiment, a CELLFREE® CD8 assay (T Cell Sciences,Inc., Cambridge, Mass.) can be used (described in U.S. Pat. No.5,006,459; see Section 8, infra). In a preferred embodiment for themeasurement of total CD4 antigen levels, anti-CD4 mAbs 8F4 and R2B7 canbe used as the capture and detection reagents, respectively, in asandwich enzyme immunoassay; in a more preferred embodiment, a CELLFREE®CD4 assay (T Cell Sciences, Inc., Cambridge, Mass.) can be used(described in International Patent Publication WO 90/04180; see Sections6 and 7, infra). In a preferred embodiment for the measurement of totalVδ1 antigen levels, anti-TCRδ mAbs TCRδ1 and δTcs1 can be used in asandwich enzyme immunoassay (see Section 9, infra). In a preferredembodiment for the measurement of total β TCR antigen levels, anti-βTCRmAbs βF1 and W4 can be used in a sandwich enzyme immunoassay (seeSection 10, infra). The foregoing antibodies have been deposited withthe ATCC, as described in Section 10, infra.

Various procedures known in the art may be used for the production ofantibodies to leukocyte marker. Such antibodies include but are notlimited to polyclonal, monoclonal, chimetic, single chain, Fab fragmentsand an Fab expression library. For the production of antibodies, varioushost animals, including but not limited to rabbits, mice, rats, etc.,may be immunized by injection with a leukocyte marker. In oneembodiment, leukocyte marker may be conjugated to an immunogeniccarrier. In another embodiment, leukocyte marker epitope, e.g., ahapten, is conjugated to a carrier. As used herein, an "epitope" is afragment of an antigen capable of specific immunoactivity, e.g.,antibody binding. Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

Monoclonal antibodies to leukocyte marker may be prepared by using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include but are not limited tothe hybridoma technique originally described by Kohler and Milstein,(1975, Nature 256:495-497), the more recent human B-cell hybridomatechnique (Kosbor et al., 1983, Immunology Today 4:72) and theEBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96). In an additionalembodiment of the invention, monoclonal antibodies specific forleukocyte marker may be produced in germ-free animals utilizing recenttechnology (PCT/US90/02545). According to the invention, humanantibodies may be used and can be obtained by using human hybridomas(Cote et al., 1983, Proc. Nat'l. Acad. Sci., U.S.A. 80:2026-2030) or bytransforming human B cells with EBV virus in vitro (Cole et al., 1985,in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96).In fact, according to the invention, techniques developed for theproduction of "chimeric antibodies" (Morrison et al., 1984, Proc. Nat'l.Acad. Sci. U.S.A. 81:6851- 6855; Neuberger et al., 1984, Nature312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing thegenes from a mouse antibody molecule specific for leukocyte markertogether with genes from a human antibody molecule of appropriatebiological activity can be used; such antibodies are within the scope ofthis invention.

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778) can be adapted toproduce a leukocyte marker-specific single chain antibody. An additionalembodiment of the invention utilizes the techniques described for theconstruction of Fab expression libraries (Huse et al., 1989, Science246:1275-1281) to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity for leukocyte marker.

Antibody fragments which contain sites specific for leukocyte marker maybe generated by known techniques. For example, such fragments includebut are not limited to: the F(ab')₂ fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the F(ab')₂fragments.

The above-described method can also be used to assay any total leukocytesurface marker, e.g., the markers described in Sections 2.1 supra. Forexample, and not by way of limitation, total CD4, total CD8, or totalTCR may be measured in the practice of this invention. In anotherembodiment, a plurality of two or more total leukocyte surface moleculesor markers can be measured. In yet a further embodiments, the relativeamount of one marker can be compared to another.

5.2. ENUMERATION OF LEUKOCYTES POSITIVE FOR A MARKER

The measurement of a total leukocyte marker according to the inventionprovides a method to enumerate the number of leukocytes that arepositive for the leukocyte marker in a sample. In another embodiment,the activity of a leukocyte marker in an assay can be compared to theactivity of a standard that comprises a known number of cells positivefor the leukocyte marker.

This aspect of the invention is based on the discovery that the totalleukocyte marker level in a sample, which is proportioned to theactivity of the leukocyte mark in an assay for the leukocyte marker,correlates directly with the number of cells positive for that marker.As shown in an Example, infra (Section 7), the correlation between thenumber of CD4+ cells determined according to the present invention andthe number of cells determined by fluorescence-activated cell sorting(FACS) has a correlation coeffecient of 0.946, which indicates thatenumeration of a leukocyte marker-positive cell by the total leukocytemarker method of the invention can be as reliable as enumeration by flowcytometry. Furthermore, as pointed out in the Summary of the Invention,supra, the present methods do not require complicated samplepreparation, careful storage conditions, or expensive analyticalequipment.

In another embodiment, the amount of leukocyte marker in the membraneand/or intracytoplasmic compartments of a sample, i.e., not includingsoluble leukocyte marker, correlates with the number of cells in thesample. As shown in an Example, Section 8 infra, the correlation betweenthe number of cells positive for a leukocyte marker and the amount ofleukocyte marker in the membrane and intracytoplasmic compartments isgood.

According to the present invention, the number of cells positive for aleukocyte marker in a sample can be determined by (1) determining thetotal amount of leukocyte marker in a sample, and (2) calculating thetotal number of cells positive for the leukocyte marker from the totalamount of leukocyte marker. In one embodiment, the calculation can bemade by extrapolating from a standard curve of total leukocyte markerversus total number of cells positive for the leukocyte marker. Inanother embodiment, a formula can be derived from a standard curve, andthe total amount of leukocyte marker plugged into the formula. In yetanother embodiment, only the activity of the assay for a total leukocytemarker is used to calculate the total number of cells positive for themarker. In this embodiment, the total amount of leukocyte marker isreflected in the relative activity in the detection assay. The method ofcalculating a total number of cells, e.g., extrapolation or a formula,remains the same.

The present invention provides simple, straightforward methods tocalculate a standard. In one embodiment, the total amount of leukocytemarker relates to the total number of cells positive for the marker. Ina more preferred embodiment, a linear regression analysis of the totalleukocyte marker to total cell number positive for the marker isperformed. The linear regression analysis can yield a straight linecurve, an estimate of error, and a formula for calculating total cellnumber from total leukocyte marker amount.

In another embodiment, the activity of the leukocyte marker in thedetergent-treated sample can be detected by immunological detectionmeans and compared with an assay of a known amount of leukocyte markerin a standard sample, and the number of cells positive for the leukocytemarker determined from the comparison. In a more preferred embodiment,the activity in an assay of the leukocyte marker in thedetergent-treated sample can be compared with the activity in an assayof the leukocyte marker in a standard containing a known number of cellspositive for the marker, and the number of cells positive for theleukocyte marker in the sample determined from the comparison.

In another embodiment, the correlation of total leukocyte marker withcell number positive for that marker can be corrected for an excessamount of leukocyte marker in the soluble or total compartment. Theamount of soluble leukocyte marker can be assayed by the sameimmunological detection means, e.g., using the same immunoassay. Theamount of soluble marker can be subtracted from the amount of totalmarker. The corrected cell-associated leukocyte marker value cancorrelate more accurately with the cell count. In a specific embodiment,the total number of CD8+ cells correlates with the total amount of CD8minus soluble CD8 (see Section 8, infra).

In preparing a standard, total cell number can be independentlydetermined by any means known in the art, e.g., FACS, immunofluorescencemicroscopy, and complement lysis assays, to mention a few. In apreferred embodiment, the independent total cell number is obtained byFACS.

A total CD4 assay may be used to estimate the member of CD4⁺ T cells ina patient sample. The estimate of the number of CD4⁺ T cells in a samplecan be used for diagnosis or monitoring of a therapeutic treatment,e.g., for AIDS patients.

In a preferred embodiment, CD8⁺ cells can be enumerated by measuring thetotal amount of CD8 antigen in the sample and subtracting the amount ofsoluble CD8 antigen in the same sample. Such an analysis yieldsinformation on both the immune system activation and the CD8⁺ cellcount.

In yet a further embodiment the total number of T cells positive for αβor γδ TCAR can be determined. Similarly, the total number of T cellspositive for subsets of Vα, Vβ, Vγ or Vδ can be determined. Thus, wholeblood samples can now be analyzed quickly, safely and reliably for thenumber of specific subsets of T cells.

5.3. KITS

The reagents necessary for the practice of the present invention can beconveniently provided in a kit. The essential elements of a kit of theinvention are a concentrated non-ionic detergent solution and a meansfor immunologically detecting the presence of an antigen.

The kit can comprise any immunological detection means known in the art,e.g, those discussed in Section 5.1., supra. Preferably theimmunological detection means are a first capture antibody specific fora leukocyte marker, and a second detectable antibody specific for theleukocyte marker. The first antibody can be provided on a solid support,such as a glass or plastic bead, membrane, a plastic stick, a microwell,a glass or plastic test tube, or other solid supports known in the artfor immunoassays. Alternatively, the first antibody can be provided forlater immobilization. The first and second antibodies can be provided asa lyophilized preparation for reconstitution, or as a concentratedsolution. The second antibody can be detectable by labeling means. In apreferred embodiment, the labeling means is an enzyme.

The detergent solution is provided in a container having sufficientvolume to hold the amount of solution necessary for the number of assayscontemplated by the kit. Preferably the detergent solution comprises twoor more non-ionic detergents. In a preferred embodiment, the detergentsolution comprises 9% TRITON® X-100 and 6% NONIDET® " p-40 in distilledwater. That particular detergent concentration is optimum with respectto an assay for both CD4 and CD8.

Preferably the kit comprises a leukocyte marker standard, either insolution or lyophilized for reconstitution. More preferably, the kitcomprises a standard comprising a known number of cells positive for theleukocyte marker of interest. Thus, the activity in an assay for theleukocyte marker in an unknown sample can be directly compared with aknown sample, and the number of cells in the unknown sample that arepositive for the leukocyte marker enumerated thereby.

The kit can further comprise a reagent reactive with label, when thesecond antibody is detectable by attachment of a label to the antibody.For example, when the label is an enzyme, the kit can provide the enzymesubstrate. The kit can also provide a dilution buffer, in finalconcentration, high concentration for dilution, or dry forreconstitution.

In yet a further embodiment, a kit provides immunological detectionmeans for more than one leukocyte antigen. In a preferred embodiment,the kit comprises immunological detection means for both CD4 and CD8.

5.4. METHODS OF DIAGNOSIS AND MONITORING OF A DISEASE 5.4.1. METHODS OFDIAGNOSIS

In another embodiment of the present invention, measurement of a totalleukocyte marker can be used to detect, diagnose or stage a disease ordisorder in a subject. The measured amount of the total leukocyte markeris compared to a baseline level, i.e., the amount of total-leukocytemarker in normal individuals. This baseline level can be the amountwhich is established to be normally present in the body fluid ofsubjects prior to the onset of disease or the amount present duringremission of disease.

Disease or disorders which may be detected, diagnosed or staged in asubject according to the present invention include but are not limitedto those listed in Table III.

                  TABLE III                                                       ______________________________________                                        DISEASES AND DISORDERS WHICH MAY BE                                           DETECTED AND/OR DIAGNOSED AND/OR                                              MONITORED IN A SUBJECT ACCORDING TO                                           THE PRESENT INVENTION                                                         ______________________________________                                        I.        Infectious Diseases Induced by virus                                Herpesvirus                                                                   Cytomegalovirus                                                               Epstein-Barr Virus                                                            HTLV-I                                                                        HTLV-III / LAV / HIV (AIDS)                                                   II.       Cancer                                                              B or T cell leukemia                                                          HTLV-I- associated adult T cell leukemia                                      B or T cell lymphoma                                                          Burkitt's lymphoma                                                            Hairy cell leukemia                                                           Sezary syndrome                                                               Hodgkin's disease                                                             Chronic lymphocytic leukemia                                                  Non-Hodgkin's lymphoma                                                        B-cell acute lymphoblastic leukemia                                           Solid tumors                                                                  III.      Autoimmune Diseases                                                 Rheumatoid arthritis                                                          Diabetes                                                                      Multiple sclerosis                                                            Systemic lupus erythematosis                                                  IV.       Organ Allograft Rejection                                           ______________________________________                                    

The methods for detecting diseases or disorders based on the number ofcells in a sample from the patient that are positive for a leukocytemarker are discussed in Section 2.3 supra and in the references citedtherein. Those methods can be practiced by the present methods of theinvention more cheaply and easily than heretofore thought possible.

In one embodiment, the amount of total CD4 antigen can be used toenumerate CD4 positive cells which in turn can be used to diagnose AIDS.In the past, the diagnosis of AIDS was determined when a patient who wasHIV-positive demonstrated CD4 positive cell counts of <500 cells/mm³ inthe presence of one or more opportunistic infections. This definition ofAIDS is expected to change to HIV-positive in the presence of <200cells/mm³ whether or not opportunistic infections are present (seeSection 2.3., supra).

Responses to viral infections can also be monitored by measuring totalCD8 levels in a patient. For example, patients infected with herpesvirus or an AIDS virus can present modified levels of total CD8. Inother embodiments, total levels can be measured in transplant patients,and used as a diagnostic indication of allograft rejection. Detection ofincreased levels of total CD8 can be associated with rheumatoidarthritis and infectious diseases such as EBV-induced mononucleosis.Detection of elevated levels of a CD8 antigen can indicate theinvolvement of significant numbers of suppressor/cytotoxic T cells witha specific pathological event, distinct from immune activation.

In yet another embodiment, the measurement of total CD4 in a cellculture can be relied on as an indication of the CD4⁺ phenotype of thelymphocytes present. For example, CD4⁺ leukemias or lymphomas can beclassified using total CD4 antigen measurements. In a furtherembodiment, the measurement of total CD4 antigen can be used to detectCD4⁺ cells and to enumerate them. Similar embodiments of the inventioninclude the measurement of total CD8 antigen to classify CD8⁺ leukemiasor lymphomas, to detect CD8⁺ cells and to enumerate them.

5.4.2. METHODS OF MONITORING

The present invention provides a method for monitoring the effect of atherapeutic treatment on a subject who has undergone the therapeutictreatment. This method comprises measuring at suitable time intervalsthe amount of a total leukocyte marker. Any change or absence of changein the amount of the total leukocyte marker can be identified andcorrelated with the effect of the treatment on the subject.

In one embodiment, total CD4 can be measured (and CD4⁺ cells enumerated)and used in the prediction of therapeutic outcome of AIDS patientsfollowing administration of therapeutic compounds such as AZT,interferon or CD4.

In another embodiment, total CD8 antigen can be measured and correlatedwith disease progression or treatment outcome.

Measurement of total T cell antigen receptor can be especially useful inmonitoring the effectiveness of treatment with agents such as T cellreceptor specific antibodies. In specific embodiments, the total TCRantigen in a specific subset of T cells expressing specific variableregions can be measured and correlated with treatment outcome.

The therapeutic treatments which may be evaluated according to thepresent invention include but are not limited to radiotherapy, drugadministration, vaccine administration, immunosuppressive orimmunoenhansive regimens, etc. The immunosuppressant regimens include,but are not limited to administration of drugs such as Cyclosporin A,chlorambucil, cyclophosphamide, or azathioprine, and anti-T cellantibody such as anti-T3 monoclonal antibody, anti-T cell antigenreceptor antibody, and anti-thymocyte globulin, etc. The immunoenhansiveregimens include, but are not limited to administration ofinterleukin-1, interleukin-2, interleukin-4 and other T cell growthfactors.

5.4.3. METHODS OF DIAGNOSIS OR MONITORING OF THERAPY BASED ON DETECTIONOF A PLURALITY OF LEUKOCYTE MARKERS

The present invention also provides for the detecting or diagnosis ofdisease, or the monitoring of treatment by measuring a plurality (atleast two) of total leukocyte markers. For example, a plurality of Tcell markers either in total form, for example but not limited to CD4and CD8, and TCAR to mention but a few, can be measured to diagnose,detect, or monitor treatment of diseases or disorders. Such diseases ordisorders include those indicated in Table III. Total marker levels canrepresent a measure of immune system function, paralleling diseasecourse or treatment efficacy. In a preferred embodiment, the prognosticindicator is the observed change over time in different marker levelsrelative to one another, rather than the absolute levels of the markerspresent at any one time. Since CD4, CD8 and TCAR are indicators of theimmune system function, they should provide a much improved measure ofthe relative health of the immune system during various stages ofdisease or disorders.

In a particular embodiment, diseases and disorders caused by HIV (thecausative agent of AIDS) infection may be monitored by measurements of aplurality of leukocyte surface markers. AIDS therapies include thetreatment of AIDS patients with drugs such as AZT(azido-deoxythymidine), γ or β interferons, and with soluble CD4, or itsfragments and derivatives. In another embodiment, the efficacy ofpotential AIDS vaccines, such as gp120 peptides can be tested bymonitoring a plurality of markers. Practitioners in AIDS therapy verymuch need a procedure that can be used to monitor the efficacy of thesetreatments or vaccines. To date, the levels of the HIV antigen p24 havenot proved sensitive enough. Total CD4 relative to total CD8 can bedetected in HIV-infected patients with different manifestations ofdisease, providing a sensitive immunoassay to monitor AIDS therapies andvaccines. The measurement of total CD4 and total CD8 is an inexpensiveand easy immunoassay format and is a valuable clinical tool forpredicting disease prognosis and treatment outcome in AIDS patients.Detection of total CD4 and CD8 according to the method of the inventionantigen provides a particularly useful way to follow HIV infection andAIDS therapy since the relative level of CD4 positive T cells decreasedramatically relative to the total number of CD8 positive cells in theprogress of AIDS etiology.

In a preferred aspect, the approach that can be taken is to determinethe levels of total CD4 and total CD8 levels in longitudinal timestudies and to compare these values with a baseline level. The baselinelevel can be either the level of the marker present in normal,disease-free individuals or the level present in a patient prior totreatment, during remission of disease, or during periods of stability.These levels can then be correlated with the disease course or treatmentoutcome.

The present invention also provides for the detection or diagnosis ofdisease or the monitoring of treatment by measuring the amounts of totalleukocyte marker and of soluble leukocyte marker in a sample andcomparing the two measurements. The change in the levels of theleukocyte markers relative to one another can be an improved prognosticindicator. In one embodiment, the level of soluble CD8 (a measure ofimmune system activation, see International Patent Publications WO87/05912 published Oct. 8, 1987 and WO 90/04180 published Apr. 19, 1990)is compared with the level of CD8 antigen obtained by subtracting theamount of soluble CD8 antigen from the amount of total CD8 antigen(e.g., the difference yields cell membrane bound plus cytoplasmic CD8,which is equivalent to cell-associated total CD8). Such a comparisongives information on the relative level of immune system activation andon changes in the number of cells in the CD-positive cell subset, bothuseful in the monitoring of disease progression or treatment. In apreferred embodiment, both the soluble and total CD8 antigen levels canbe determined using one immunoassay configuaration, but with differentsample treatment before assay.

The instant invention will be further clarified by the followingExamples, which are provided as purely exemplary of the invention andare not intended as limiting of the invention.

6. EXAMPLE: A TOTAL CD4 ANTIGEN ASSAY

The present example demonstrates the development of an assay thatcorrelates total CD4 values with the number of CD4⁺ T cells in a sample.

6.1. MATERIALS AND METHODS 6.1.1. SAMPLE PREPARATION

Samples. Patient samples included 26 sero-positive HIV-infectedindividuals and 13 healthy adult volunteers. Blood was obtained byvenipuncture into a blood collection tube.

Sample Treatment. Prior to assay, whole blood samples were treated asfollows.

Step 1:

100 μl of anticoagulated blood was removed from the blood collectiontube and mixed with 20 μl of concentrated 6× detergent (6% TRITON®X-100, 6% NONIDET® "p-40 in distilled water) in a 12×75 mm glass tube.This mixture was then incubated for one minute at room temperature.After one minute the treated sample was either used in a CD4 immunoassayor stored at -70° C. until assayed. Samples stored at -70° C. wereallowed to thaw at room temperature before use.

Step 2:

Prior to assaying the treated samples in a CD4 immunoassay, the sampleswere diluted as indicated in the results section with sample buffer (1%bovine serum albumin, phosphate buffered saline, 0.25% NONIDET®" p-40and 0.01% thimerosal) to dilute the concentration of detergent. Totalsample volumes added to the immunoassay were maintained at 50 μl.

6.1.2. CD4 IMMUNOASSAY

Total CD4 antigen was measured in a CD4 specific immunoassay (seeInternational patent publication WO 87/05912 published Oct. 8, 1987)involving a one-step, three hour format using microtiter plates that hadbeen precoated with capture antibody overnight. Briefly, each well of a96 well microtiter well plate was coated with 100 μl of murine anti-CD4coating antibody in phosphate-buffered saline (PBS) overnight at 4° C.Any remaining protein-binding sites were blocked with 300 μl per well ofblocking buffer (0.5% casein, 0.008% NONIDET®" P-40, 0.005% EDTA in PBS)for 2 hours at 37° C. The wells were washed three times with 350 μl perwell of wash buffer (PBS, ph 7.4, with 0.05% Tween 20). After aspiratingthe final wash buffer from the wells, 50 μl of horseradish peroxidase(HRP) conjugated murine monoclonal anti-human CD4 antibody (in PBS with15% FCS and 0.15% NONIDET®" p-40) and 50 μl of sample or standard wereadded to each well of the microtiter plate in duplicate. The combinedvolume of sample and HRP conjugated antibody was 100 μl. Samples andantibodies were incubated for 3 hours at room temperature. After washingthe plate as described above, 100 μl of OPD substrate (OPD tablets,BioDesign Intl, Catalogue #A45104T, dissolve 1 tablet in 4 ml citratebuffer-peroxide, BioDesign Intl, Kennebunkport, Me. Catalog numberA45105B) was added to all the wells and incubated for 30 minutes at roomtemperature. At the end of this last incubation, 50 μl of 2 N H₂ SO₄ wasadded to each well to stop the reaction and absorbance of each well wasread at 490 nm. Results were plotted as values obtained for each sampleat O.D. 490 against total CD4⁺ cells/mm³ or sample volume. Correlationcoefficients were calculated using linear regression analysis.

Whole blood samples from normal and HIV-infected patients were assayedfor absolute CD4⁺ T-cells/mm³ using the formula:

    Abs. CD4.sup.+ T cells/mm.sup.3 =WBC×% Lymphocyte×% CD4.sup.+ T cells.

WBC (White blood cell count) was determined using a hemacytometer. %Lymphocyte was determined by a differential count, and %CD4⁺ T cellswere determined using the Ortho cytoflurograph II and Leu-3a (anti-CD4)fluorescein-conjugated antibody (Becton Dickinson, Mountain View,Calif.)

6.2. EXPERIMENTAL RESULTS

Experiment 1: Measurement of total CD4 antigen in whole blood. Thisstudy was conducted to determine whether detergent treatment of wholeblood would yield reliable measurements of total CD4 antigen in a CD4immunoassay. Samples of whole blood were treated with a 6× detergentsolution using a ratio of 20 μl detergent to 100 μl anticoagulated wholeblood. Serial dilutions were made of these samples to produce a seriesof samples containing decreasing amounts of total CD4 antigen. Thesesamples were then analyzed in the CD4 assay to determine which dilutionsproduced values that were linear with sample dilution. As the assayconfiguration became saturated with excess CD4, the optical densityvalues representing total CD4 values became nonlinear.

The results of this experiment can be seen in FIG. 1. Total CD4preparations made from three healthy volunteers were treated withconcentrated non-ionic detergent, and diluted in serial 1:2 increments(from 1/2 to 1/32). Sample volumes were maintained at 50 μl by dilutioninto sample buffer. Linear regression analysis demonstrated that samplescontaining less than 20 μl and preferably 5-10 μl of detergent treatedwhole blood were optimal for total CD4 measurement as they fell withinthe linear range. Samples containing greater than 20 μl of whole bloodsaturated the assay and total CD4 could not be accurately determined.

Experiment 2: Specificity of the total CD4 antigen measurement. Theability of an anti-CD4 antibody to block the total CD4 signal detectedin whole blood samples of three normal controls is presented in TableIV. The assay was run as described above, except that before adding theHRP-conjugated murine anti-CD4 monoclonal antibody to the wells, 5 μl ofthe unconjugated murine anti-CD4 monoclonal antibody used in the coatingprocedure was added to the wells, followed by the HRP-conjugatedantibody. Samples were diluted with sample diluent to a final volume of50 μl. The unconjugated antibody was able to block total CD4 detectionby 50%, 51% and 51% in samples #1, 2, and 3, respectively, at the levelof competing antibody used. This demonstrates the specificity of theassay to detect total CD4. Other proteins released during detergentlysis of the whole blood samples (such as hemoglobin) did not interferewith the assay.

TABLE IV

                  TABLE IV                                                        ______________________________________                                        SPECIFICITY OF TOTAL CD4 DETECTION IN                                         DETERGENT TREATED WHOLE BLOOD                                                 O.D. 495                                                                      sample #                                                                             10 μl*  10 μl + anti-CD4**                                                                       % inhibition                                  ______________________________________                                        1      0.433      0.216         50%                                           2      0.928      0.472         51%                                           3      0.833      0.422         51%                                           ______________________________________                                         *10 μl of detergent treated whole blood prepared from normal               uncoagulated whole blood                                                      **5 μl of the antiCD4 coating antibody from the CD4 assay was also         added to compete for binding.                                            

Experiment 3: Enumeration of cell number using total CD4 values. Thisstudy was done to determine whether the measurement of total CD4 levelsin treated whole blood samples would correlate with absolute CD4positive cell numbers. Data in FIG. 2 demonstrate a statisticallysignificant correlation between total CD4 measured in whole bloodsamples and the total number of CD4⁺ cells/mm³ of blood (r=0.904).Samples measured in this assay contained 2.5 μl of detergent treatedwhole blood diluted to a final volume of 50 μl from a total of fivenormal controls. The normal range of CD4⁺ cells/mm³ is 800-1000cells/mm³. The normal values in this assay appear lower than usual dueto the fact that the WBC count used to determine the number of CD4cells/mm³ was performed on the uncoagulated blood samples three daysafter the CD4 assay was run. The relationship between total CD4 detectedin the assay and the total CD4⁺ cells/mm³ for each individual wasdetermined by linear regression analysis and a statistically significantr value of 0.904 was obtained. This indicates that the measurement oftotal CD4 antigen can be used to directly enumerate CD4 positive cells.

Experiment 4: Enumeration of CD4⁺ cells in HIV-infected and normalindividuals using measurements of total CD4 antigen. After theproperties of the total CD4 method were established using healthydonors, further study was conducted to establish a relationship betweenthe total CD4 as measured in the ELISA to the total number of CD4⁺ Tcells in HIV infected individuals.

The total CD4 antigen values of HIV-infected individuals and normalswere determined using the total CD4 method on two separate occasions.The result of these experiments are presented in FIGS. 3 and 4. Ten μlof whole blood samples diluted to a total sample volume of 50 μl fromeither the disease group or control group were used to determine thetotal CD4 antigen values. The total CD4⁺ cells/mm³ cells was determinedon fresh uncoagulated blood samples on the day of the CD4 assay.

Both FIGS. 3 and 4 demonstrate a statistically significant correlationbetween total CD4 antigen measured in the assay and the total CD4⁺cells/mm³ for both the HIV-infected group (single squares) and thecontrol group (double squares) (r=0.860 and 0.867 for FIGS. 3 and 4,respectively). Thus, the measurement of total CD4 antigens fromdetergent treated whole blood accurately reflects the absolute number ofCD4⁺ T-cells in whole blood for both normal and HIV infectedindividuals.

6.3. DISCUSSION

As described herein, we have developed a method that measures the levelof total CD4 antigen in a patient sample. Whole blood samples weredetergent treated and assayed using a CD4 immunoassay in a one-step,three hour format. It was demonstrated that total CD4 antigen could bedetected in whole blood samples of normal individuals by a simpledetergent lysis step and CD4 immunoassay. Optimal detection could bemeasured using 10 μl or less of detergent treated whole blood samples.Accurate and reproducible measurements can be detained from 2.5 μl to 5μl of whole blood. The detection of total CD4 antigen was specific as amurine anti-CD4 monoclonal antibody was capable of blocking the totalCD4 antigen signal when added to the assay format. The release of otherproteins during detergent treatment of whole blood did not interferewith the specificity of the assay. It was further demonstrated that thetotal CD4 antigen values obtained in the ELISA correlated with the totalCD4⁺ T cells/mm³ in the whole blood of normal donors (r=0.9).

A comparison between the total CD4 antigen obtained in the CD4 ELISA andthe total number of CD4⁺ T Cells/mm³ of whole blood yielded astatistically significant correlation in both HIV-infected individualsand normals. This comparison was determined on two separate occasionsusing two different groups of HIV-infected individuals and normals. Bothcomparisons yielded statistically significant correlations between totalCD4 antigen values and total CD4⁺ T cells/mm³ (r=0.86 and 0.87). Sincethe measurement of total CD4 antigen accurately reflects the number ofCD4 positive cells, the total CD4 method can be used to enumerate CD4positive cells and to determine the numbers of CD4 positive cells indisease diagnosis or monitoring of treatment, etc.

7. EXAMPLE: ENUMERATION OF CD4 POSITIVE LYMPHOCYTES IN PERIPHERAL BLOODBY THE TOTAL CD4 METHOD

This Example outlines further development and characterization of thetotal CD4 method for enumeration of CD4 positive cells. The methodconsists of 3 steps: treatment of samples with concentrated detergent,dilution of the concentrated detergent lysate and CD4 immunoassay.

7.1. MATERIALS AND METHODS

Samples: Ninety-five samples of whole blood were collected in EDTAcontaining vacutainer tubes and mixed thoroughly. Samples were analyzedfor white blood cell count and differential and CD4⁺ lymphocytes by flowcytometry.

Sample Treatment: Samples were treated as described in section 6, exceptfor the following:

Step 1:

1 volume of concentrated detergent solution was added to 5 volumes offresh EDTA whole blood, followed by gentle mixing. Usually 100 μl ofwhole blood was treated with 20 μl of detergent solution. A comparisonof detergents of various concentrations indicated that the best resultswere obtained using a combination of detergents such as TWEEN-20 withTRITON®" X-100 or TRITON®" X-100 with NP40. Any of the three detergentsalone was not as effective. The detergent treated sample (100 μl sampleplus 20 μl concentrated detergent) yielded to a final combined detergentconcentration of 2.5%. The optimal detergent solution for measuring bothtotal CD4 and total CD 8 using the same concentrated detergent solutionwas 9% TRITON®" X100 and 6% NONIDET®" (15% total in the concentrateddetergent solution) which corresponded to 1.5% TRITON®" X100 and 1%NONIDET®" (2.5% total) in the treated sample. In other experiments, thefinal concentration of the detergents in the treated sample wassimilarly effective over a range of at least 1-2% TRITON®" p-40 X100combined with 1-1.5% NONIDET®".

Other methods of lysis, solubolization or disruption of cells includinghypotonic treatment with distilled water, sonication, or addition ofdetergent at lower final concentrations (e.g. one detergent at 1%concentration in the treated sample) were tested and found noteffective.

Step 2:

After 5 minutes, 100 μl of treated sample was diluted with 400 μl ofdilution buffer to yield a 1:5 dilution. Fifty μl of this diluted sample(about 10 μl of whole blood) was used for each assay well. The assayconfiguration allowed whole blood volumes of 5-25 μl to be measured,since the resulting values fell within the range of the standard curve.Blood volumes above or below these amounts yielded values that felloutside the range of the standard curve. The importance of step 2 is thedilution of the detergent concentration in the treated sample to prevent"stripping" in the following assay and loss of signal. Samplescorresponding to 50 μl of whole blood performed poorly in the CD4immunoassay because the resulting values were outside the assay range,and because the detergent concentration was disruptive to the antibodysandwich formation or stability.

Assay. Samples were stored at -70° C. prior to assay. The samples wereassayed in the one-step sandwich immunoassay as described in section 6.Total CD4 values were determined from a standard curve. Standards weresoluble, recombinant CD4 antigen prepared by removing the transmembraneregion of the CD4 gene, followed by expression of the truncated gene inmammalian cells and recovery of recombinant CD4 antigen in thesupernatant of the cell culture (see International Patent PublicationsWO88/01304 and WO89/02922).

7.2. EXPERIMENTAL RESULTS

Experiment 5: A standard curve for the total CD4 antigen method wasgenerated using culture supernatant containing recombinant CD4 receptor(see FIG. 5). For the assay configuration used, the standard range wasdetermined to be 0 to 200 units/μl with a correlation coefficient of0.999.

Experiment 6: Specificity of the assay for the CD4 antigen. Bloodsamples were drawn from 4 normal volunteers and treated as follows: Asample from each was analyzed for white blood cell count anddifferential (the percentage of WBCs that were lymphocytes; see theformula in Section 6.1.2). The rest of the sample was divided; one-halfwas depleted of CD4⁺ cells by absorption with anti-CD4 coated Dynabeads(Dynal, Inc. Great Neck, N.Y.) while the other half was untreated. Thedepleted and untreated samples were again divided and one fractionanalyzed by flow cytometry while the other was treated using the totalCD4 method. CD4 positive cell number was calculated from the white bloodcell count, lymphocyte differential, and percentage of CD4 cells by flowcytometry using the formula described in Section 6.1.2. The comparisonof CD4 positive cell number to total CD4 antigen is given in Table V.

                  TABLE V                                                         ______________________________________                                        Specificity of the Total CD4 Antigen Assay for CD4 Antigen                    Untreated Fractions                                                                              Depleted Fractions                                              Flow       Total CD4  Flow     Total CD4                                      Cytometry  Antigen    Cytometry                                                                              Antigen                                   #    cells/μl                                                                              units/μl                                                                              cells/μl                                                                            units/μl                               ______________________________________                                        1    71         320        0        7                                         2    934        445        0        11.5                                      3    1109       345        40       15                                        4    912        395        45       15                                        ______________________________________                                    

Both the flow cytometry and the total CD4 methods indicated the removalof CD4 positive cells from all depleted fractions indicating that theassays were specific for the CD4 receptor.

Experiment 7: Assay dilution and linearity. This study was done tofurther verify assay sensitivity. Whole blood samples from normalvolunteers were collected in EDTA. A and a portion of each was depletedof CD4 positive cells to serve as the sample diluent in order to keepthe sample matrix as normal as possible. Samples were prepared neat andat 1/2, 1/4 and 1/8 dilutions with concentrated detergent; and run inthe CD4 immunoassay. Table VI shows the results of this experiment on 6different samples. Values are presented as raw data as run in the assay.The zero value represents the assay value obtained for the depletedportion used as sample diluent. This value is included in thecalculations of expected values.

                  TABLE VI                                                        ______________________________________                                        Dilution and Linearity of Assay                                                                Units      Units  Percent                                    Sample  Dilution Detected   Expected                                                                             Recovery                                   ______________________________________                                        585     Neat     64         64     100                                                1:2      35         35.5   99                                                 1:4      24         21     114                                                1:8      14         14     100                                                0        7                                                            586     Neat     89         89     100                                                1:2      58         51     114                                                1:4      34         31     110                                                1:8      22         21     105                                                0        11.5                                                         593     Neat     79         79     100                                                1:2      38         41     91                                                 1:4      26         22     119                                                1:8      22         24     92                                                 0        16                                                           598     Neat     63         63     100                                                1:2      40         39     103                                                1:4      30         27     111                                                1:8      26         21     124                                                0        15                                                           599     Neat     69         69     100                                                1:2      36         42     86                                                 1:4      28         28     100                                                1:8      22         22     100                                                0        15                                                           600     Neat     65         65     100                                                1:2      36         40     90                                                 1:4      27         27     100                                                1:8      21         21     100                                                0        15                                                           ______________________________________                                    

These results indicate that normal whole blood samples dilute linearlyin the assay. The assay background is <20 units. Neat normal samplesfall in the middle of the assay range, so dilution of even 1:2 should berarely necessary.

Experiment 8: Cell enumeration using the total CD4 method. This studyconfirmed that it was possible to enumerate CD4 positive cells in wholeblood by the total CD4 method. Both flow cytometry and total CD4 antigenassays were run by Maryland Medical Laboratory on normal and abnormalblood specimens that were less than 24 hours old. Flow cytometry wasperformed on a Coulter Epics Flow Cytometer; the CD4 immunoassay wasread using a Molecular Devices VMAX ELISA reader. Samples with low CD4positive cell counts were selected from HIV positive specimens. Cellswere dually stained with anti-CD4 and anti-CD2 antibodies in order tomake sure that the cells counted by Flow were CD4 positive T cells. Atotal of 95 samples (46 normal and 49 abnormal) were assayed for whiteblood cell number, lymphocyte differential, and percent CD2⁺ CD4⁺ cellsby flow cytometry and for the amount of CD4 by the total CD4 antigenmethod. The number of CD4.sup. + T lymphocytes was calculated from thecell count, differential and flow data and compared to the data obtainedfrom the total CD4 method by linear regression. The distribution of thetwo types of samples was:

    ______________________________________                                        46 Normals                                                                    CD4+ T cell Number  500-1600 cells/μl                                      total CD4 antigen   222-600 units/ml                                          49 Abnormals                                                                  CD4+ T cell Number   1-690 cells/μl                                        total CD4 antigen    48-295 units/ml                                          ______________________________________                                    

The results of the linear regression analysis are shown in FIG. 6, whereunits/ml from tile total CD4 antigen method are compared to cells/μlfrom the flow analysis. The slope of the line (0.26) and Y-intercept(89.7) define the linear regression curve equation Y=0.26×+89.7, wherethe total CD4 antigen units/ml is calculated to be 0.26 times the totalnumber of cells/μl plus 89.7. The correlation coefficient for thecomparison is 0.946, which indicates that enumeration of CD4 positivecells via the total CD4 antigen method is as reliable as the sameenumeration by flow cytometry.

7.3. DISCUSSION

The quantitation of CD4 receptor protein in treated samples of wholeblood has been shown to be equivalent to flow cytometry for theenumeration of CD4 positive T lymphocytes (r=0.946). For theconfiguration of the assay used, sensitivity is 100 units/ml or 50cells/μl, which allows the total CD4 antigen method to be used over awide range (50-1600 cells/μl) of samples. In the 95 samples tested, thepossible presence of CD4 positive monocytes did not interfere with theassay results. Since only 10 μl of whole blood is needed per test, thetotal CD4 method will be useful for detecting CD4⁺ T cells in samplesthat are difficult to obtain. The total CD4 method can be run inlaboratories without access to flow cytometry. The method is simple andcan be competed in less than 4 hours. Samples can be treated quickly andassayed immediately or treated and stored frozen for batch testing. Suchbatch testing of longitudinal patient samples following a course oftreatment is not subject to the errors obtained with flow cytometrymeasurements where samples are run separately on different days. Thedetergent treatment step of the total CD4 method also provides the addedbenefit of inactivating enveloped viruses which increases operatorsafety.

8. EXAMPLE: ENUMERATION OF CD8 POSITIVE CELLS BY THE TOTAL CD8 METHOD8.1. MATERIALS AND METHODS

Samples: Patient samples included 49 sero-positive HIV-infectedindividuals and 46 healthy adult volunteers with CD8⁺ cell countsranging from 28 to 2983 cells/μl.

Sample Treatment: Whole blood samples were prepared as described inSection 7, supra. Two hundred μl of concentrated detergent solution (9%TRITON®" X100 and 6% NONIDET®" P-4 were added to 1 ml of whole blood.Following gentle mixing, the sample was diluted 1:5 (50 μl treatedsample plus 200 μl diluent) with diluent before assay. Treated sampleswere stored at -70° C. prior to assay.

Assay. Total CD8 was measured in detergent treated whole blood in aCELLFREE® CD8 immunoassay (T cell Sciences, Inc.) using a one-step,three hour format. Briefly, each well of a 96 well microtiter plate wascoated with 100 μl of the murine anti-CD8 coating antibody inphosphate-buffered saline overnight at 4° C. The coating buffer wasremoved and 300 μl of blocking buffer (0.5% casein, 0.008% NONIDET®"P-40, 0.005% EDTA in PBS) were added per well and incubated for 2 hoursat 37° C. After washing the wells three times with 400 μl of wash buffer(PBS, pH 7.4, with 0.05% Tween 20), 50 μl of horseradish peroxidase(HRP) conjugated murine monoclonal anti-human CD8 antibody (10% FCS,0.025% thimerosal, 0.01% gentamicin, 0.05% tween in Tris BufferedSaline, TBS) were added to all wells except those used as blanks. Fiftyμl of CD8 standard or treated whole blood was added to 200 μl of diluent(1% bovine serum albumin, phosphate buffered saline, 0.25% NONIDET®"P-40 and 0.01% thimerosal) and 50 μl of this were added to theappropriate microtiter wells. The plate was covered with plate sealerand incubated for three hours at 20° C. with shaking at 150 rpms. Afterwashing the plate as described above, 100 μl of OPD substrate were addedto all the wells, and the plate was incubated at 20° C. for 30 minutes.Fifty μl of 2N H₂ SO₄ were added to all wells to stop the reaction andabsorbance was read at 490 nm. A standard curve (correlation >0.999;range 0-2300 units/μl; assay background <100 units) was constructed byplotting O.D. against the concentration of the standards. Theconcentration of total CD8 in the whole blood samples was thendetermined from the standard curve. Correlation coefficients werecalculated using linear regression analysis.

Whole blood samples from normal and HIV-infected patients were assayedfor absolute CD8⁺ T-cells/mm³ using the formula:

    Abs. CD8.sup.+ T-cells/mm.sup.3 =WBC×% lymphocyte×% CD8.sup.+ T cells.

WBC (white blood cell count) was determined using a hemacytometer. %Lymphocyte was determined by a differential count, and %CD8⁺ T-cellswere determined using the Ortho cytoflurograph II and Leu-2a (anti-CD8,) fluorescein conjugated antibody (Becton Dickinson, Mountain View,Calif.)

8.2. EXPERIMENTAL RESULTS

Experiment 1. The specificity and linearity of the assay for CD8 weredetermined using similar depletion and dilution studies as described inSection 7. Total CD8 antigen values were obtained from whole blood of 43normal and 43 HIV-infected, sero-positive individuals and compared tothe total number of CD8⁺ T-cells/mm³ of whole blood. Table VIIsummarizes the population statistics for the normal and abnormal samplesby flow cytometry and the total CD8 method. The normal samples have alinear correlation of r=0.772 and the HIV⁺ samples have a linearcorrelation of r=0.718.

                  TABLE VII                                                       ______________________________________                                        AVERAGE RANGE FOR 45 NORMAL PATIENT SAMPLES                                   26 patients were male                                                                          19 patients were female                                      ages ranged from 23-69 years                                                                   ages ranged from 18-65 years                                 ______________________________________                                                                                Total                                                                         CD8                                   Samples                                                                              WBC*     % Lymph   % CD8* #CD8*  units/ml                              ______________________________________                                        average                                                                              6.48     31        23     483    949                                   2 STD  3.04     10        14     181    343                                   High   10.2     41        42.3   898     2300+                                Low    4.0      22        8.2    173    423                                   ______________________________________                                        AVERAGE RANGE FOR 49 HIV.sup.+  PATIENT SAMPLES                               45 patients were male                                                                          4 patients were female                                       ages ranged from 24-65 years                                                                   ages ranged from 21-43 years                                 ______________________________________                                                                                Total                                                                         CD8                                   Samples                                                                              WBC*     % Lymph   % CD8* #CD8*  units/ml                              ______________________________________                                        average                                                                              4.66     36        55     871    1368                                  2 STD  5.76     26        28     1050   1244                                  High   19.9     61        79.5   2983    2300+                                Low    1.0      0.2       12.7   28      364                                  ______________________________________                                    

FIG. 7 demonstrates a statistically significant correlation betweentotal CD8 antigen measured in the assay and the total number of CD8⁺cells (r=0.75). In this study of 86 individuals, it was found that whentotal CD4 values were normal, so were total CD8 values. As CD4 valuesdeclined in HIV⁺ patients, the total CD8 values varied widely. Sampleswith low CD4 (<180 units/ml) and low CD8 values generally had low whiteblood cell counts as well. The HIV⁺ patients had some of the highest CD8counts, perhaps to replace their diminished CD4⁺ T cells.

Experiment 2. Although it would be possible to enumerate CD8 positivecells using the total CD8 method of experiment 1 (r=0.75), thisquantitation was not as accurate as the enumeration of CD4 positivecells by the total CD4 method (r=0.946). Since activation of CD8positive cells results in the release of large amounts of soluble CD8antigen, it is likely that the levels of sCD8 were affecting thecorrelation of total CD8 antigen levels with CD8 positive cell counts.To test this hypothesis, total CD8 antigen and soluble CD8 antigen weremeasured in 30 normal and 11 cancer (mainly melanoma and lymphoma) wholeblood samples. Total CD8 antigen measurements were made as describedabove with the detergent treatment, dilution and immunoassay, andsoluble CD8 levels were determined directly by immunoassay of plasmapreparations. The same CD8 immunoassay was used for both the total CD8antigen and soluble CD8 antigen. Since one volume of plasma (samplesize) has twice the amount of plasma as an equivalent sized sample ofwhole blood, the soluble CD8 amounts in plasma were divided by two. Theamount of cell associated CD8 (membrane bound plus cytoplasmic) wasdetermined by subtracting the soluble levels of CD8 (divided by two)from the amount of total CD8 antigen. The results of this analysis areshown in FIG. 8. Linear regression analysis of this data gave animproved correlation coefficient of 0.859 as compared to 0.75 for totalCD8 antigen alone. Cell enumeration could be more accurately quantitatedfrom the equation:

    Y (cells/mm3)=1.1×(units/ml total CD8 antigen)+7.4.

8.3. DISCUSSION

As described herein, we have developed a total CD8 method that measuresthe level of total CD8 antigen in a patient sample. Whole blood sampleswere detergent treated, diluted, and assayed using a CD8 immunoassay ina one-step, three hour format. Total CD8 antigen values obtained in thetotal CD8 method correlated with total CD8⁺ T-cell number (r=0.75) inwhole blood. A significant improvement in the ability to enumerate CD8⁺cells in whole blood was found when the level of soluble CD8 antigen(released from activated cells) was subtracted from the level of totalCD8 antigen (r=0.859). Monitoring the relationship of soluble and totalantigen provided improved information on cell count and immuneactivation. Thus, the total CD8 assay alone, or in combination withother soluble or total marker assays, can be used to monitor the immuneprofile of patients.

9. EXAMPLE: ENUMERATION OF TCAR POSITIVE CELLS BY TOTAL TCR METHODS

Total TCAR β Experiments. An assay to detect total T cell antigenreceptor (TCAR) β chain or total Vβ5 specific TCAR chain was performedusing detergent treated whole blood samples in a total TCR method.Briefly, wells in a 96 well plate were coated with 5 μg/ml of coatingantibodies which consisted of either a negative control antibody (δTCS1which is specific for the Vδ1 region of the γδ T cell receptor), or W76which recognizes the constant region of the β chain (Cβ) or a Vβ5specific monoclonal antibody such as W112 (Tian et al., 1989, FASEB J.3:A486 Abstr). A horseradish peroxidase conjugated (HRP)-βF1 antibody,which recognizes a different epitope of the β chain constant region (Cβ)than W76, was used as the detection antibody. Whole blood lysates wereprepared as described in Section 7, supra. The assay format was similarto that described in U.S. Pat. No. 4,845,026, issued Jul. 4, 1989 andentitled "Assay Systems for Detecting Cell-Free T Cell Antigen ReceptorRelated Moleucles and Clinical Utilities of the Assays." In addition, aone-step, three hour format as described in Section 7, supra, wasperformed with similar results. Twenty-five μl of whole blood lysate wasdiluted in 75 μl of sample diluent for a starting dilution of 1:4. O.D.values from the negative control wells were subtracted from all valuesobtained (negative control wells using δTCS1 as the coating antibodyaveraged 0.10 and blank wells which had no antibody averaged 0.095).

The results of the total TCAR assay can be seen in FIG. 9. TCAR β chainwas optimally detected at dilutions of 1:4 and 1:8. Vβ5 specific TCARwas detected at low levels (1:16 dilution) as well. Lower levels of Vβ5were expected, since the Vβ5 positive subset of cells represents only asmall portion of all β TCAR positive T cells. The assay demonstratedgood specificity as the background O.D. was no higher when the controlantibody δTCS1 was used as the coating antibody as compared to O.D.sobtained from blank wells which contain no coating antibody (0.100 and0.095 respectively).

These data demonstrate that a total TCAR method can be used to detectthe total amount of TCR β chain in a whole blood sample by using twomonoclonal antibodies which recognize different regions of the constantregion of the β chain. The total amount of β chain is an indication ofthe total amount of β positive T cells in the sample. Furthermore,specific subsets such as the Vβ5 family, which represent only 1-5% ofthe total TCAR β chains in normal blood, can be detected.

Total TCAR Vδ1 Experiments. The treatment of samples was similar to thatof section 7 with a concentrated detergent solution of 9% TRITON®" X-100and 6% NONIDET®" P-40 in 1× PBS that was added to whole blood at a ratioof 200 μl concentrated detergent solution to 1 ml whole blood. Sampleswere then diluted 1:2 prior to immunoassay. The immunoassay procedurewas similar to the CD4 or CD8 immunoassay except with antibody TCRδ1 ascapture antibody and δTCS1 as detection antibody. The TCAR δ positivecell line PEER was used as a positive control. A standard curve wasgenerated with the units arbitraily assigned based upon a PEER cellsample preparation stored at -70° C. and assigned a value of 20,000units. The specificity of the assay for TCAR δ chain was determinedusing an antibody-affigel stripping procedure. Affigel 10 was coupled toeither BSA, anti-CD8 antibody 4C9 (an isotype control antibody), δTCS1,TCRδ1, Vδ2 or Vγ2 at 1 mg/ml concentration. A 50/50 slurry was made ofeach sample. Three hundred μl of a detergent treated PEER cell samplewas added to 50 μl of each antibody-affigel slurry. The sample/gels wereincubated for 1 hour at 4° C. The samples were then centrifuged for 5minutes at 10 K. The supernatant fluid was removed and added to 50 μlmore of the antibody-affigel slurries. The samples/gels were incubatedfor another hour at 4° C. This process was repeated for a total of threesample/affigel incubations. After the last centrifuge spin, thesupernatants were removed and run in the assay as samples. Thesupernatants were not diluted 1:2 prior to assay, because the PEERsample was diluted 1:25 in sample diluent buffer before theantibody-affigel stripping. Samples that were not stripped withantibody-affigel were kept at 4° C. while the other samples were beingstripped. The only two antibodies to completely strip the TCR δreactivity from the samples were the TCAR δ chain specific antibodiesδTCR1 and TCSδ1, indicating that the assay was specific for TCR δ chain.When 8 samples were run in the total TCAR δ chain method, total TCAR δranged from 55 to 108 units (FIG. 10). These all fell at the lower endof the standard curve.

The success of these total TCAR methods for measuring total β or total δTCR antigen amounts can be extended to total Vα, total γδ, total αβ orto total Vγ, and related subset assay procedures as well. Detection ofthe total amount of the above β and δ antigens is useful in cell typingand in correlating specific V region expression to disease state ortreatment outcome.

10. DEPOSIT OF HYBRIDOMAS

The following hybridoma cell lines, producing the indicated monoclonalantibody, have been deposited with the American Type Culture Collection,Rockville, Md. and have been assigned the listed accession numbers:

    ______________________________________                                        Accession                                                                     Hybridoma Number                                                                             Monoclonal Antibody                                                                           --                                             ______________________________________                                        Cell line 4C9  4C9 (anti-CD8)  HB 9340                                        Cell line 5F4/7B12                                                                           5F4/7B12 (anti-CD8)                                                                           HB 9342                                        Cell line 8F4  8F4 (anti-CD4)  HB 9843                                        Cell line R2B7 R2B7 (anti-CD4) HB 9842                                        Cell line 8A3.31                                                                             8A3.31 (βF1)                                                                             HB 9283                                        Cell line δTCS1                                                                        δTCAR3 = δTCS1                                                                    HB 9578                                        Cell line W112 W112 (Vβ5) HB 9927                                        Cell line W4F.5B                                                                             W4F.5B (anti-Cβ)                                                                         HB 9282                                        Cell line 5A6.E9                                                                             TCRδ1     HB 9772                                        ______________________________________                                    

The present invention is not to be limited in scope by the cell linesdeposited since the deposited embodiments are intended as singleillustrations of one aspect of the invention and any cell lines whichare functionally equivalent are within the scope of this invention.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and accompanying drawings. Suchmodifications are intended to fall within the scope of the appendedclaims.

Various references are cited herein, the disclosures of which areincorporated by reference herein in their entireties.

What is claimed is:
 1. A method for determining the total amount of a leukocyte marker in an original sample suspected of having the leukocyte marker, which original sample contains cells and a biological fluid in which cells are obtained, comprising:(a) adding a non-ionic detergent to the original sample to form a detergent treated sample in an amount sufficient to release any intracellular and membrane bound markers; (b) allowing the detergent treated sample to lyse the cells therein; (c) diluting the detergent treated sample by at least two fold; and (d) determining the amount of leukocyte marker in the detergent treated sample and calculating the total amount of leukocyte marker in the original sample therefrom.
 2. The method according to claim 1 in which the non-ionic detergent comprises more than one non-ionic detergent.
 3. The method according to claim 1 or claim 2 in which the non-ionic detergent is selected from the group consisting of TRITON®" X-100, NONIDET®" P-40, Tween-20 and CHAPS.
 4. The method according to claim 1 in which the nonionic detergent comprises "TRITON®" X-100 at a concentration of 1% to 2% and "NONIDET®" P-40 at a concentration of 1% to 2%, which concentrations of "TRITON®" X-100 and "NONIDET®" are their respective concentrations in the detergent treated sample.
 5. The method according to claim 1 in which the concentration of non-ionic detergent in the detergent-treated sample is a virus-inactivating concentration of non-ionic detergent.
 6. The method according to claim 1 in which the non-ionic detergent is allowed to lyse the cells for at least about 1 minute.
 7. The method according to claim 1 in which the detergent-treated sample is stored at about -20° C. or less after step (b).
 8. The method according to claim 7 in which the detergent-treated sample is stored at about -70° C. or less.
 9. The method according to claim 1 in which the original sample is selected from the group consisting of whole blood, blood cells, saliva, urine, synovial fluid, pleural effusions, tumor and tissue infiltrates, amniotic fluid, spinal fluid, cranial fluid, tissue, and tissue culture fluid.
 10. The method according to claim 9 in which the sample is whole blood.
 11. The method according to claim 1 in which the determination of step(d) is accomplished by an immunoassay selected from the group consisting of radio immunoassay, enzyme-linked immunoassay, "sandwich" immunoassay, immunoradiometric assay, fluorescence immunoassay and protein A immunoassay.
 12. The method according to claim 11 in which the immunoassay detection means is a "sandwich" immunoassay.
 13. The method according to claim 12 in which the sandwich immunoassay employs a polyclonal antibody.
 14. The method according to claim 13 in which the sandwich immunoassay further employs a monoclonal antibody.
 15. The method according to claim 1 in which the leukocyte marker is selected from the group consisting of CD4, CD8 and T cell antigen receptor.
 16. The method according to claim 14 or 15 in which the leukocyte marker is selected from the group consisting Of CD4, CD8 and T cell antigen receptor.
 17. The method according to claim 1 in which the leukocyte marker is CD4 and in which the determination of step (d) is accomplished by a sandwich immunoassay employing a first antibody 8F4, deposited with the ATCC and assigned accession no. HB 9843, and a second antibody R2B7, deposited with the ATCC and assigned accession no. HB
 9842. 18. The method according to claim 1 in which the leukocyte marker is CD8 and in which the determination of step (d) is accomplished by a sandwich immunoassay employing a first antibody 4C9, deposited with the ATCC and assigned accession no. HB 9340, and a second antibody 5F4/7B12, deposited with the ATCC and assigned accession no. HB
 9342. 19. The method according to claim 1 in which the leukocyte marker is a β T cell antigen receptor, and in which the determination of step (d) is accomplished by a sandwich immunoassay employing a first antibody selected from the group consisting of VβS-specific antibody W112, deposited with the ATCC and assigned accession no. HB9927, and Cβ-specific antibody W4F.5b, deposited with the ATCC and assigned accession no. HB 9282, and a second antibody 8A3.31 (βF1), deposited with the ATCC and assigned accession no. HB
 9283. 20. The method according to claim 1 in which the leukocyte marker is a δ T cell antigen receptor, and in which the determination of step (d) is accomplished by a sandwich immunoassay employing a first antibody TCFδ1, deposited with the ATCC and assigned accession no. HB 9772, and a second antibody δTCS1, deposited with the ATCC and assigned accession no. HB
 9578. 21. A method for determining the total amount of a leukocyte marker in an original sample suspected of having the leukocyte marker which original sample contains cells and the biological fluid in which the cells are obtained, comprising:(a) adding a non-ionic detergent to the original sample to form a detergent-treated sample such that the final concentration of the non-ionic detergent in the detergent treated sample is 2% to 4%: (b) allowing the detergent to lyse the cells therein; (c) diluting the detergent treated sample by at least 2-fold; and (d) determining the amount of leukocyte marker in the detergent treated sample and calculating the total amount of leukocyte marker in the original sample therefrom.
 22. A method for diagnosing, detecting or staging a disease comprising detecting in a sample from a patient suspected of having the disease the total amount of a leukocyte marker according to the method of claim 1, 2, 4, 12 or 21 and comparing the amount so detected to the amount of the total leukocyte marker in normal individuals, a difference in amounts indicating a disease state.
 23. The method according to claim 22 in which the leukocyte marker is selected from the group consisting of CD4, CD8 and T cell antigen receptor.
 24. The method according to claim 23 in which the leukocyte marker is CD4 and the disease is selected from the group consisting of acquired immunodeficiency syndrome (AIDS), AIDS related complex, and CD4⁺ leukemia or lymphoma.
 25. The method according to claim 23 in which the leukocyte marker is CD8 and the disease is selected from the group consisting of AIDS, transplantation rejection, rheumatoid arthritis, and infectious disease.
 26. A method for diagnosing, detecting or staging a disease in a patient suspected of having the disease comprising:(a) measuring in an original sample from the patient the total amount of at least two leukocyte markers according to the method of claim 1, 2, 4, 12 or 21; (b) determining the ratio or ratios of the amounts of the leukocyte markers measured in step (a); and (c) comparing the ratio or ratios of the leukocyte markers determined in step (b) to the same ratio or ratios in a normal individual, a difference in the ratios indicating a disease state.
 27. The method of claim 26 in which the leukocyte markers are CD4 and CD8 and the disease is AIDS.
 28. A method for monitoring the effect of a therapeutic treatment for a disease comprising detecting in a sample from a patient undergoing therapeutic treatment for the disease the total amount of a leukocyte marker according to the method of claim 1, 2, 4, 12 or 21 and comparing the amount so detected to the amount of the total leukocyte marker in normal individuals, a difference in amounts indicating a change in the disease state.
 29. The method according to claim 28 in which the leukocyte marker is selected from the group consisting of CD4, CD8 and T cell antigen receptor.
 30. The method according to claim 29 in which the leukocyte marker is CD4 and the disease is selected from the group consisting of acquired immunodeficiency syndrome (AIDS), AIDS related complex, and CD4⁺ leukemia or lymphoma.
 31. The method according to claim 29 in which the leukocyte marker is CD8 and the disease is selected from the group consisting of AIDS, transplantation rejection, rheumatoid arthritis, and infectious disease.
 32. A method for monitoring the effect of a therapeutic treatment for a disease in a patient undergoing therapeutic treatment for the disease comprising:(a) measuring in a sample from the patient the total amount of at least two leukocyte markers according to the method of claim 1, 2, 4, 12 or 21; (b) determining the ratio or ratios of the amounts of the leukocyte markers measured in step (a); and (c) comparing the ratio or ratios of the leukocyte markers determined in step (b) to the same ratio or ratios in a normal individual, a difference in the ratios indicating a change in the disease state.
 33. The method of claim 32 in which the leukocyte markers are CD4 and CD8 and the disease is AIDS, and in which an increase in the ratio of CD4 to CD8 positive leukocytes in the patient relative to the patient at an earlier time is indicative improvement of the disease.
 34. A method for enumerating the total number of leukocytes that are positive for a CD4 leukocyte marker in an original sample, which original sample contains cells and a biological fluid in which cells are obtained, comprising:(a) adding a non-ionic detergent to the original sample to form a detergent treated sample such that the final concentration of the non-ionic detergent in the detergent treated sample is 2% to 4%; (b) allowing the detergent to lyse the cells therein; (c) diluting the detergent treated sample by at least two fold; and (d) detecting the total amount of the CD4 leukocyte marker in the detergent treated sample; and (e) calculating the total amount of leukocytes positive for the CD4 leukocyte marker from the total amount of CD4 leukocyte marker detected in step (d).
 35. The method according to claim 34 in which the non-ionic detergent comprises more than one non-ionic detergent.
 36. The method according to claim 34 or 35 in which the non-ionic detergent is selected from the group consisting of "TRITON®" X-100, "NONIDET®" P-40, Tween-20 and CHAPS.
 37. The method according to claim 36 in which the non-ionic detergent comprises "TRITON®" X-100 at a concentration of 1% to 2% and "NONIDET®" P-40 at a concentration of 2%, which concentrations of "TRITON®" X-100 and "NONIDET®" P-40 are their respective concentrations in the detergent-treated sample.
 38. The method according to claim 37 in which the non-ionic detergent is allowed to lyse the cells for at least about 1 minute.
 39. The method according to claim 38 in which the detergent-treated sample is stored at about -20° C. or less after step (b).
 40. The method according to claim 39 in which the original sample is selected from the group consisting of whole blood, blood cells, saliva, urine, synovial fluid, pleural effusions, tumor and tissue infiltrates, amniotic fluid, spinal fluid, cranial fluid, tissue, and tissue culture fluid.
 41. The method according to claim 34 in which the original sample is whole blood.
 42. The method according to claim 35 in which the original sample is whole blood.
 43. The method according to claim 36 in which the original sample is whole blood.
 44. The method according to claim 37 in which the original sample is whole blood.
 45. The method according to claim 38 in which the original sample is whole blood.
 46. The method according to claim 39 in which the original sample is whole blood.
 47. The method according to claim 40 in which the original sample is whole blood.
 48. The method according to claim 40 in which the determination of step (d) is accomplished by an immunoassay selected from the group consisting of radioimmunoassay, enzyme-linked immunoassay, "sandwich" immunoassay, immunoradiometric assay, fluorescence immunoassay and protein A immunoassay.
 49. The method according to claim 34 in which the immunoassay is a "sandwich" immunoassay.
 50. The method according to claim 42 in which the sandwich immunoassay employs a polyclonal antibody.
 51. The method according to claim 42 in which the sandwich immunoassay further employs a monoclonal antibody.
 52. The method according to claim 44 in which the immunoassay detection means is a sandwich immunoassay employing a first antibody 8F4, deposited with the ATCC and assigned accession no. HB 9843, and a second antibody R2B7, deposited with the ATCC and assigned accession no. HB
 9842. 53. The method according to claim 34 further comprising:(i) separately measuring the amount of soluble leukocyte marker in the original sample; (ii) subtracting the amount of soluble leukocyte marker in the original sample from the amount of total leukocyte marker in the original sample determined in step (d); and, (iii) in step (e) calculating the number of cells positive for the leukocyte marker in the original sample from the remainder in step (ii).
 54. A method for enumerating the total number of leukocytes that are positive for a CD8 leukocyte marker in an original sample, which original sample contains cells and the biological fluid in which the cells are obtained comprising:(a) adding a non-ionic detergent to the original sample to form a detergent treated sample such that the final concentration of the non-ionic detergent in the detergent treated sample is 2% to 4%; (b) allowing the detergent to lyse the cells therein; (c) diluting the detergent treated sample by at least two fold; and (d) detecting the total amount of the CD8 leukocyte marker in the detergent treated sample; and (e) calculating the number total amount of leukocytes positive for the CD8 leukocyte marker from the total amount of CD8 leukocyte marker detected in step (d).
 55. The method according to claim 54 in which the non-ionic detergent comprises more than one non-ionic detergent.
 56. The method according to claim 54 or 55 in which the non-ionic detergent is selected from the group consisting of "TRITON®" X-100, "NONIDET®" P-40, Tween-20 and CHAPS.
 57. The method according to claim 56 in which the non-ionic detergent comprises "TRITON®" X-100 at a concentration of 1% to 2% and "NONIDET®" P-40 at a concentration of 1% to 2%, which concentrations of "TRITON®" X-100 and "NONIDET®" P-40 are their respective concentrations in the detergent-treated sample.
 58. The method according to claim 57 in which the non-ionic detergent is allowed to lyse the cells for at least about 1 minute.
 59. The method according to claim 58 in which the detergent-treated sample is stored at about -20° C. or less after step (b).
 60. The method according to claim 59 in which the original sample is selected from the group consisting of whole blood, blood cells, saliva, urine, synovial fluid, pleural effusions, tumor and tissue infiltrates, amniotic fluid, spinal fluid, cranial fluid, tissue, and tissue culture fluid.
 61. The method according to claim 54 in which the original sample is whole blood.
 62. The method according to claim 55 in which the original sample is whole blood.
 63. The method according to claim 56 in which the original sample is whole blood.
 64. The method according to claim 57 in which the original sample is whole blood.
 65. The method according to claim 58 in which the original sample is whole blood.
 66. The method according to claim 59 in which the original sample is whole blood.
 67. The method according to claim 60 in which the original sample is whole blood.
 68. The method according to claim 60 in which the determination of step (d) is accomplished by an immunoassay selected from the group consisting of radioimmunoassay, enzyme-linked immunoassay, "sandwich" immunoassay, immunoradiometric assay, fluorescence immunoassay and protein A immunoassay.
 69. The method according to claim 68 in which the immunoassay is a "sandwich" immunoassay.
 70. The method according to claim 69 in which the sandwich immunoassay employs a polyclonal antibody.
 71. The method according to claim 70 in which the sandwich immunoassay further employs a monoclonal antibody.
 72. The method according to claim 71 in which the immunoassay is a sandwich immunoassay employing a first antibody 4C9, deposited with the ATCC and assigned accession no. HB 9340, and a second antibody 5F4/7B12, deposited with the ATCC and assigned accession no. HB
 9342. 73. The method according to claim 54 further comprising:(i) separately measuring the amount of soluble leukocyte marker in the original sample; (ii) subtracting the amount of soluble leukocyte marker in the original sample from the amount of total leukocyte marker in the original sample determined in step (d); and (iii) in step (e) calculating the number of cells positive for the leukocyte marker in the original sample from the remainder in step (ii).
 74. A method for enumerating the total number of leukocytes that are positive for a T cell antigen receptor leukocyte marker in an original sample, which original sample contains cells and the biological fluid in which the cells are obtained, comprising:(a) adding a non-ionic detergent to the original sample to form a detergent treated sample such that the final concentration of the non-ionic detergent in the detergent treated sample is 2% to 4%; (b) allowing the detergent to lyse the cells therein; (c) diluting the detergent treated sample by at least two fold; and (d) detecting the total amount of the T cell antigen receptor leukocyte marker in the detergent treated sample; and (e) calculating the total amount of leukocytes positive for the T cell antigen receptor leukocyte marker from the total amount of the T cell antigen receptor leukocyte marker detected in step (d).
 75. The method according to claim 74 in which the non-ionic detergent comprises more than one non-ionic detergent.
 76. The method according to claim 74 or 75 in which the non-ionic detergent is selected from the group consisting of "TRITON®" X-100, "NONIDET®" P-40, Tween-20 and CHAPS.
 77. The method according to claim 76 in which the non-ionic detergent comprises "TRITON®" X-100 at a concentration of 1% to 2% and "NONIDET®"P-40 at a concentration of 1% to 2%, which concentrations of "TRITON®" X-100 and "NONIDET®"P-40 are their respective concentrations in the detergent-treated sample.
 78. The method according to claim 77 in which the non-ionic detergent is allowed to lyse the cells for at least about 1 minute.
 79. The method according to claim 78 in which the detergent-treated sample is stored at about -20° C. or less after step (b).
 80. The method according to claim 79 in which the original sample is selected from the group consisting of whole blood, blood cells, saliva, urine, synovial fluid, pleural effusions, tumor and tissue infiltrates, amniotic fluid, spinal fluid, cranial fluid, tissue, and tissue culture fluid.
 81. The method according to claim 74 in which the original sample is whole blood.
 82. The method according to claim 75 in which the original sample is whole blood.
 83. The method according to claim 76 in which the original sample is whole blood.
 84. The method according to claim 77 in which the original sample is whole blood.
 85. The method according to claim 78 in which the original sample is whole blood.
 86. The method according to claim 79 in which the original sample is whole blood.
 87. The method according to claim 80 in which the original sample is whole blood.
 88. The method according to claim 80 in which the immunoassay is selected from the group consisting of radioimmunoassay, enzyme-linked immunoassay, "sandwich" immunoassay, immunoradiometric assay, fluorescence immunoassay and protein A immunoassay.
 89. The method according to claim 88 in which the immunoassay is a "sandwich" immunoassay.
 90. The method according to claim 89 in which the sandwich immunoassay employs a polyclonal antibody.
 91. The method according to claim 89 in which the sandwich immunoassay further employs a monoclonal antibody.
 92. The method according to claim 91 in which the T cell antigen receptor is a β T cell antigen receptor, and in which the immunoassay is a sandwich immunoassay employing a first antibody selected from the group consisting of Vβ5-specific antibody W112, deposited with the ATCC and assigned accession no. HB 9927, and Cβ-specific antibody W4F.5B, deposited with the ATCC and assigned accession no. HB 9832, and a second antibody 8A3.31 (βF1), deposited with the ATCC and assigned accession no. HB
 9283. 93. The method according to claim 91 in which the T cell antigen receptor is a β T cell antigen receptor, and in which the immunoassay is a sandwich immunoassay employing a first antibody TCRδ1, deposited with the ATCC and assigned accession no. HB 9772, and a second antibody δTCS1, deposited with the ATCC and assigned accession no. HB
 9578. 94. The method according to claim 74 further comprising:(i) separately measuring the amount of soluble leukocyte marker in the original sample; (ii) subtracting the amount of soluble leukocyte marker in the original sample from the amount of total leukocyte marker in the original sample determined in step (d); and (iii) in step (e) calculating the number of cells positive for the leukocyte marker in the original sample from the remainder in step (ii).
 95. A method for diagnosing, detecting or staging a disease in a patient suspected of having the disease comprising enumerating the number of leukocytes that are positive for a leukocyte marker in a sample from the patient according to the method of claim 34, 54 or 74 and comparing the number of leukocytes with the number of leukocytes found in a normal individual, a difference in the numbers indicating a disease state.
 96. The method according to claim 95 in which the leukocyte marker is CD4 and the disease is selected from the group consisting of acquired immunodeficient syndrome (AIDS), AIDS related complex, and CD4⁺ leukemia or lymphoma.
 97. The method according to claim 95 in which the leukocyte marker is CD8 and the disease is selected from the group consisting of AIDS, transplantation rejection, rheumatoid arthritis, and infectious disease.
 98. A method of diagnosing, detecting or staging a disease in a patient suspected of having the disease comprising:(a) enumerating the number of leukocytes that are positive for at least two leukocyte markers in a sample from the patient according to the method of claim 34, 54 or 74, (b) determining the ratio or ratios of leukocytes positive for each of the leukocyte markers enumerated in step (a); and (c) comparing the ratio or ratios of leukocytes determined in step (b) to the ratio or ratios in a normal individual, a difference in the ratios indicating a disease state.
 99. The method of claim 98 in which the leukocyte markers are CD4 and CD8 and the disease is AIDS.
 100. A method for monitoring the effect of a therapeutic treatment for a disease in a patient undergoing therapeutic treatment for the disease comprising enumerating the number of leukocytes that are positive for a leukocyte marker in a sample from the patient according to the method of claim 34, 54, or 74 and comparing the approximate number of leukocytes with the number of the leukocytes found in normal individual, a difference in the number indicating a change in the disease state.
 101. The method according to claim 100 in which the leukocyte marker is CD4 and the disease is selected from the group consisting of acquired immunodeficiency syndrome (AIDS), AIDS related complex, and CD4⁺ leukemia or lymphoma.
 102. The method according to claim 100 in which the leukocyte marker is CD8 and the disease is selected from the group consisting of AIDS, transplantation rejection, rheumatoid arthritis, and infectious disease.
 103. A method of monitoring the effect of a therapeutic treatment for a disease in a patient undergoing therapeutic treatment for the disease comprising:(a) enumerating the number of leukocytes that are positive for at least two leukocyte markers in a sample from the patient according to the method of claim 34, 59 or 74; (b) determining the ratio or ratios of leukocytes positive for each of the leukocyte markers enumerated in step (a); and (c) comparing the ratio or ratios of leukocytes determined in step (b) to the ratio or ratios in the patient at an earlier time difference in the ratios indicating a change in the disease state.
 104. The method of claim 103 in which the leukocyte markers are CD4 and CD8 and the disease is AIDS, and in which an increase in the ratio of CD4 to CD8 positive leukocytes in the patient relative to the patient at an earlier time is indicative of improvement of the disease.
 105. A kit for enumerating the number of leukocytes that are positive for a CD4 leukocyte marker in a sample comprising:(a) a concentrated non-ionic detergent solution comprising 9% "TRITON®" X-100 and 6% "NONIDET®" P-40 in distilled water; and (b) an immunoassay employing a first antibody 8F4, deposited with the ATCC and assigned accession no. HB 9843, and a second antibody R2B7, deposited with the ATCC and assigned accession no HB
 9853. 106. A kit for enumerating the number of leukocytes that are positive for a CD8 leukocyte marker in a sample comprising:(a) a concentrated non-ionic detergent solution comprising 9% "TRITON®" X-100 and 6% "NONIDET®" P-40 in distilled water; and (b) an immunoassay employing a first antibody 4C9, deposited with the ATCC and assigned accession no. HB 9340, and a second antibody 5F4/7B12, deposited with the ATCC and assigned accession no. HB
 9353. 107. A kit for enumerating the number of leukocytes that are positive for a β T cell antigen receptor leukocyte marker in a sample comprising:(a) a concentrated non-ionic detergent solution comprising 9% "TRITON®" X-100 and 6% "NONIDET®" P-40 in distilled water; and (b) an immunoassay employing a first antibody selected from the group consisting of Vβ5-specific antibody W112, deposited with the ATCC and assigned accession no. HB 9927, and Cβ-specific antibody W4F.5B, deposited with the ATCC and assigned accession no. HB 9832, and a second antibody 8A3.31 (βF1), deposited with the ATCC and assigned accession no. HB
 9283. 108. A kit for enumerating the number of leukocytes that are positive for a delta T cell antigen receptor leukocyte marker in a sample comprising:(a) a concentrated nonionic detergent solution comprising 9% "TRITON®" X-100 and 6% "NONIDET®" P-40 in distilled water; and (b) an immunoassay employing a first antibody TCRδ1, deposited with the ATCC and assigned accession no. HB 9772, and a second antibody δTCS1, deposited with the ATCC and assigned accession no. HB
 9578. 